Compositions and methods for treating papillomavirus-infected cells

ABSTRACT

By virtue of the present invention, there is provided methods and compositions for interfering with the proliferation of cells infected and/or transformed by papillomaviruses. The processes and compositions of this invention may be used to treat any mammal, including humans. According to this invention, mammals are treated by the pharmaceutically acceptable administration of an E2 ad/db  protein, either directly or by gene transfer techniques, to reduce the symptoms of the specific papillomavirus-associated disease, or to prevent their recurrence.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/362,012filed Jul. 27, 1999, now U.S. Pat. No. 6,432,926, which is acontinuation-in-part application of U.S. application Ser. No. 08/677,206filed on Jul. 9, 1996, now abandoned. The entire contents of theaforementioned applications and all other patents, patent applications,and references cited throughout this specification are herebyincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Papillomaviruses (PV) have been linked to widespread, serious humandiseases, especially carcinomas of the genital and oral mucosa. Tens ofmillions of women suffer from human papilloma virus (HPV) infection ofthe genital tract. Significant number of these women eventually developcancer of the cervix. It has been estimated that perhaps twenty percent(20%) of all cancer deaths in women worldwide are from cancers which areassociated with HPV. As many as 90% of all cervical cancer maybe linkedto HPV.

Papillomaviruses also induce benign, dysplastic and malignanthyperproliferations of skin and mucosal epithelium (see, for example,Mansur and Androphy, (1993) Biochim Biophys Acta 1155:323-345; Pfister(1984) Rev. Physiol. Biochem. Pharmacol. 99:111-181; and Broker et al.(1986) Cancer Cells 4:17-36, for reviews of the molecular, cellular, andclinical aspects of the papillomaviruses).

HPV's are a heterogeneous group of DNA tumor viruses associated withhyperplastic (warts, condylomata), pre-malignant and malignant lesions(carcinomas) of squamous epithelium. Almost 70 HPV types have beenidentified, and different papillomavirus types are known to causedistinct diseases, c.f., zur Hausen, (1991) Virology 184:9-13; Pfister,(1987) Adv. Cancer Res., 48:113-147; and Syrjanen, (1984) Obstet.Gynecol. Survey 39:252-265. HPVs can be further classified either highrisk (such as HPV type 16 [HPV-16] and HPV-18) or low risk (e.g, HPV-6and HPV-11) on the basis of the clinical lesions with which they areassociated and the relative propensity for these lesions to progress tocancer. For example, HPV types 1 and 2 cause common warts, and types 6and 11 cause warts of the external genitalia, anus and cervix. HPV's canbe isolated from the majority of cervical cancers, e.g., approximately85 to 90% of human cervical cancers harbor the DNA of a high-risk HPV.Types 16, 18, 31 and 33 are particularly common; HPV-16 is present inabout 50 percent of all cervical cancers.

The biological life cycle of the papillomaviruses appears to differ frommost other viral pathogens. These viruses are believed to infect thebasal or germ cells of the epithelium. Rather than proceeding to a lyticinfection in which viral replication kills the cell, viral DNAtranscription and replication are maintained at very low levels untilhigher strata of the epithelium are achieved. There, presumably inresponse to differentiation-specific signals, viral transcriptionaccelerates, DNA synthesis begins and virion assemble occurs.

In HPV-positive genital cancers, the viral genomes are transcriptionallyactive, and two viral genes, E6 and E7, are invariably expressed. Thehigh-risk HPVs encode two oncoproteins, E6 and E7, whose expression canextend the life span of squamous epithelial cells, which are a normalhost cell for the papillomavirus. E6 and E7 together can result in theefficient immortalization of primary human cells (Hawley-Nelson et al.,(1989) EMBO J., 8:3905-3910; Münger et al., (1989) J. Virol.,63:4417-4421; Watanabe et al., (1989) J. Virol., 63:965-969). E6 and E7are expressed in HPV-positive cervical cancer-derived cell lines(Schneider-Gädicke et al., (1986) EMBO J., 5:2285-2292; Schwarz et al.,(1985) Nature, (London) 314:111-114; Smotkin et al., (1986) Proc. Natl.Acad. Sci. USA, 83:4680-4684). Furthermore, although many geneticchanges have occurred in cervical carcinoma cells, the continuedexpression of the viral oncoproteins is necessary since expression ofantisense E6/E7 RNA results in decreased cell growth (vonKnebel-Doeberitz et al., (1988) Cancer Res., 48:3780-3785). Similar tothe transforming proteins of the other small DNA tumor viruses, simianvirus (SV40) and adenovirus, the transforming properties of the E6 andE7 oncoproteins appear to be due at least in part to their capacity tofunctionally inactivate the p53 and the retinoblastoma (pRB) tumorsuppressor proteins. The E6 proteins of HPV-16 and HPV-18 can complexand cause ubiquination-dependent degradation of p53 (Wemess et al.,(1990) Science, 248:76-79; Schiffaer et al., Cell 75:495-505 (1993)).The high-risk HPV E7 proteins bind pRB more efficiently than the E7proteins of low-risk HPVs (Barbosa et al., (1990) EMBO J., 9:153-160;Dyson et al., (1989) Science, 243:934-937; Münger et al., (1989) EMBOJ., 8:4099-4015). It is believed that the functional inactivation ofboth p53 and pRB, and related regulatory pathways, by E6 and E7 areimportant steps in cervical carcinogenesis.

One characteristic of HPV-related carcinogenic progression is thefrequent integration of the viral genome into the human chromosome inthe cancer cells in a manner that results in the loss of expression ofthe viral E2 gene but maintains high levels of E6/E7 expression (Dürstet al., (1985) J. Gen. Virol., 66:1515-1522; Jeon et al., (1995) Proc.Natl. Acad. Sci. USA, 92:1654-1658). The product of the E2 open readingframe plays an important role in the complex transcriptional pattern ofthe HPV's. The E2 transcriptional activation protein (“the E2 protein”)is a trans-acting factor that activates transcription through specificbinding to cis-acting E2 enhancer sequences in viral DNA (Androphy etal., (1987) Nature, 324:70-73), and has been shown to induce promoterexpression in a classical enhancer mechanism (Spalholz et al., (1985)Cell 42:183-91). The E2 gene product exerts trans-regulatory effects inthe upstream regulatory region (“LCR”) of the viral genome, disruptionof E2 is thought to alter regulation of expression of E6 and E7 genes.

As with other transcription factors, the functions of the E2 proteinsappear to be localized in discrete domains (Giri et al., (1988) EMBO J.,7:2923-29). The E2 amino terminus encompasses the transcriptionalactivation domain and binding site for the papillomavirus E1 replicationprotein. The E2 C-terminal domain is well conserved among thepapillomaviruses, and contains the dimerization and DNA bindingactivities of E2. This domain sponsors sequence-specific interactionwith DNA containing the sequence ACC(G)NNNN((C)GGT and represses thepapillomavirus early promoter that drives expression of E6 and E7 (e.g.,the P97 promoter of HPV16 and the P105 promoter of HPV 18). This is dueto the position of E2 binding sites within the promoter: two of the fourE2 binding sites within the P97 and P105 promoters immediately flank theTATA box and promoter proximal SP1 sites of these promoters, renderingthem inaccessible to needed transcription factors.

The upstream regulatory region (or long control region (LCR)) is foundimmediately 5′ to the early genes of bovine papilloma viruses (BPV's)and other papillomaviruses. The LCR contains cis-acting regulatorysignals, including an origin of DNA replication and several promotersthat function in early transcription. The LCR also contains enhancerelements that activate transcription from the URR promoters andheterologous promoters (Sousa et al., (1990) Biochemica et BiophysicaActa 1032: 19-37).

The E2 enhancer elements are conditional, in that they stimulatetranscription only when activated by a protein encoded by the E2 openreading frame (Romanczuk et al., (1990) J. of Virol. 64:2849-2859). Geneproducts from the E2 gene include the full-length transcriptionalactivator E2 protein and at least two truncated versions of the E2protein BPV1 that function as transcriptional repressors.Transcriptional activation and repression of viral genes by E2 geneproducts constitute critical regulatory circuits in papillomavirus geneexpression and DNA replication (reviewed in McBride et al., (1991) J.Biol. Chem. 266:18411-18414). Within the LCR, transcriptional regulationby the E2 protein depends on its direct binding to the nucleotidesequence 5′ACC(G)NNNN(C)GGT3′ (SEQ ID NO:9) (Androphy et al., supra;Dartmann et al., (1986) Virology, 151:124-30; Hirochika et al., (1987)J. Virol, 61:2599-606; P. Hawley-Nelson et al., (1988) EMBO J.,7:525-31; McBride et al., (1988) EMBO J., 7:533-39; McBride et al., J.of Biol. Chemistry 266:18411-1844 (1991); Demeret et al. J. Virol.71:9343-9349 (1997); Desaintes et al. EMBO 16:504-514 (1997); Thierry etal. New Biol. 10:4431-4437 (1990); and Bernard et al. J. Virol.63:4317-4324 (1989)).

European patent application 302,758 refers to the use of modified formsof E2 protein that bind to, and block, E2 binding sites onpapillomavirus DNA without resulting in trans-activation. Thatapplication also refers to repression of E2 transcriptional activationthrough the use of DNA fragments that mimic E2 binding sites, and thusbind with E2 trans-activators, making them unavailable for binding to E2sites on the viral DNA.

U.S. Pat. No. 5,219,990 describes the use of E2 trans-activationrepressors which interfere with normal functioning of the nativefull-length E2 transcriptional activation protein of the papillomavirus.However, the E2 trans-activation repressors of the '990 patent areproteins that dimerize with the full-length native E2 protein to forminactive heterodimers, thus interfering with the formation of activehomodimers comprising full-length native E2 polypeptides and therebyrepressing papillomavirus transcription and replication. The E2trans-activation repressors are described as fragments of the E2polypeptide in which the dimerization function has been separated fromits DNA binding function, e.g., the E2 trans-activation repressorsincludes at least the dimerization region, but less than the DNA bindingdomain, of the E2 polypeptide.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a method of treating, e.g.,lessening the severity or preventing the reoccurrence of, apapillomavirus-induced condition. In general, the subject methodcomprises administering to an animal, e.g. a human, infected with apapillomavirus a pharmaceutical preparation comprising a therapeuticallyeffective amount of either (i) an E2_(ad/db) polypeptide or (ii) a geneconstruct for expressing the E2_(ad/db) polypeptide. As described infurther detail below, the E2_(ad/db) polypeptide includes a DNA bindingdomain and a transcriptional activation domain derived from one or moreE2 proteins. The E2 polypeptide or gene construct is formulated in thepharmaceutical preparation for delivery into PV-infected cells of theanimal.

In preferred embodiments, the subject method is used to treat a humanwho is infected with a human papillomavirus (HPV), particularly a highrisk HPV such as HPV-16, HPV-18, HPV-31 and HPV-33. However, treatmentof low risk HPV conditions is also specifically contemplated.

In certain preferred embodiments, the DNA binding and transcriptionalactivation domains of the E2_(ad/db) polypeptide have amino acidsequences corresponding to an E2 protein(s) from an HPV, includingespecially, an E2 protein from a high risk HPV. The DNA binding domainand transcriptional activation domain of the E2 polypeptide can be onecontiguous polypeptide chain, or in those embodiments where the E2protein is directly formulated into the therapeutic composition, the DNAbinding and transcriptional activation domain portions of thetherapeutic E2 polypeptide can be provided as two separate peptidechains which have been chemically cross-linked, e.g., other than by aamide bond. The E2_(ad/db) polypeptide can be a full length E2 protein,e.g., also including a hinge region sequence or the like, or can lackother E2 peptide sequences except for the DNA binding andtranscriptional activation domains. The E2_(ad/db) polypeptide may bederived from any species, e.g., human, bovine, rabbit, and from anypapillomavirus subtype. In a preferred embodiment the E2_(ad/db) has analteration, for example, a E39A substitution or an altered hinge region,e.g., a deletion of residues corresponding to BPV E2_(Δ220-309).

The subject method can be used to inhibit pathological progression ofpapillomavirus infection, such as preventing or reversing the formationof warts, e.g. Plantar warts (verruca plantaris), common warts (verrucaplana), Butcher's common warts, flat warts, genital warts (condylomaacuminatum), or epidermodysplasia verruciformis; as well as treatingpapillomavirus-infected cells which have become, or are at risk ofbecoming, transformed and/or immortalized, e.g. cancerous, e.g. alaryngeal papilloma, a focal epithelial, a cervical carcinoma.

Another aspect of the present invention relates to a pharmaceuticalpreparation comprising a therapeutically effective amount of arecombinant transfection system for ameliorating apapillomavirus-induced condition in a subject. For instance, thetransfection system, which is for gene therapy, includes a geneconstruct having a nucleic acid encoding an E2_(ad/db) polypeptide andoperably linked to a transcriptional regulatory sequence for causingexpression of the E2 polypeptide in eukaryotic cells. The gene constructis provided in a gene delivery composition for delivering the geneconstruct to a papillomavirus infected cells and causing the cell to betransfected with the gene construct. For example, the gene deliverycomposition can be, e.g., a recombinant viral particle, a liposome, apoly-cationic nucleic acid binding agent, or a gene therapy vectorderived from, e.g., a retrovirus, adeno-associated virus, or adenovirus.

Yet another aspect of the invention relates to a pharmaceuticalpreparation comprising a therapeutically effective amount of anE2_(ad/db) polypeptide, formulated in the pharmaceutical preparation fordelivery into PV-infected cells of an animal. In preferred embodiments,the polypeptide is formulated as a liposome.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No.: 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates the growth suppression by the fulllength BPV E2 protein, but not E2-TR. The gross domain structure of thepapillomavirus E2 proteins is also illustrated.

FIG. 2 is a map of an illustrative E2-encoding adenoviral vectorsuitable for gene therapy.

FIGS. 3A and 3B illustrate the results of mutational analysis of E2proteins. FIG. 3A shows that the E2 hinge region is not required forgrowth suppression. FIG. 3B shows that the E2 transactivation domain,and a functional E2 DNA binding domain are both required for growtharrest. The properties of the various E2 proteins are also listed

FIG. 4 shows a schematic of various HPV and BPV E2 polypeptides and therelative activity of these polypeptides in viral DNA replication (DNAREP), repression of viral transcription (TXN), and suppression ofpapillomavirus-mediated cell growth (COLONIES); AD, activation domain;HG, hinge region; DB, DNA-binding domain. Mutants marked in bold, i.e.,BPV E2_(Δ220-309) and HPV_((E39A)), show a desired therapeutic responsein all three assays, i.e., no maintenance of viral replication,repression of viral gene expression, and suppression of viral-mediatedcell proliferation.

DETAILED DESCRIPTION OF THE INVENTION

The papillomaviruses (PV) are infectious agents that can cause benignepithelial tumors, or warts, in their natural hosts. Infection withspecific human papillomaviruses (HPV) is also associated with humanepithelial malignancies, including malignancies of the uterine cervix,genitalia, skin and, though less frequently, other sites.

The analysis of the temporal expression of mRNAs and the nucleotidesequence of human and animal papillomavirus (PV) genomes has revealedoverall structural similarities in their genetic organization. There areat least seven open reading frames (ORFs) in all PVs, and differentialRNA splicing provides the capability to produce several additionalproteins. The genes that are required for viral replication aretypically designated with the prefix “E” (for “early”), being expressedbefore the “L” or “late” genes. The products of the papillomavirus E2open reading frame play a key role in the regulation of the viral cycle,affecting both transcription and replication. For instance, the E2transcriptional activation protein (herein the “E2 protein”) is atrans-acting factor that activates transcription through specificbinding to cis-acting E2 enhancer sequences. The 410 amino acidpapillomavirus E2 protein has been shown to induce promoter expressionin a classical enhancer mechanism (Spalholz et al. Cell (1985)42:183-91). The E2 protein appears to provide both positive and negativefeedback loops for viral gene expression.

As with other transcription factors, the functions of the E2 proteinappear to be localized to discrete modular domains (Giri et al. (1988)EMBO J. 7:2823-2829). The C-terminal domain of the E2 polypeptide isresponsible for recognition of E2 binding sites on viral DNA, and isaccordingly referred to herein as the “DNA binding domain”. TheN-terminal domain of the E2 polypeptide is responsible fortranscriptional activation following binding of the protein to viralDNA, and is referred to as the “transactivation domain”.

In bovine papillomavirus models, and in some human papillomaviruses, atleast two N-terminally truncated E2 proteins occur naturally and act asnative repressors. It has been experimentally confirmed in vitro thattruncated forms of E2 proteins which retain their ability to bind DNAbut do not trans-activate, are competitive inhibitors oftrans-activation-competent E2 polypeptides (Lambert et al. (1987) Cell50:69-78; Stenlund et al. (1990) Genes Dev. 4:123-136; and Choe et al.(1989) J. Virol. 63:1743-1755;McBride et al. (1991) J. Bio. Chem.266:18411-18414.

As described in the appended examples, we have discovered that HPV E2proteins which retain both transactivation and DNA binding domains(“E2_(ad/db)” proteins, e.g., BPV E2_(Δ220-309) and HPV E2_((E39A))) arecapable of inhibiting cell growth of HPV-infected and/or HPV-transformedcells. On the other hand, contrary to certain teachings in the art, weobserved that growth of both HPV-infected and HPV-transformed cells wasnot apparently inhibited by the E2 transcriptional repressor (E2-TR)which represents the DNA binding domain of the E2 protein nor byVP16-E2. This result was somewhat surprising because previous studiesfound that N-terminally deleted forms of both the HPV and BPV forms ofthe E2 protein were able to transcriptionally repress the E6/E7 promoter(Thierry et al., (1991) New Biol., 3:90-100). The data presented belowsuggests that the mechanism for E2-mediated growth suppression mayinvolve more complex mechanisms or that the E2 DNA binding domain isnot, in and of itself, an effective repressor of the HPV promotersintegrated into the host chromosome.

Progression of high-risk HPV lesions to cervical cancer is almostinvariably associated with integration of the viral genome withdisruption of the E1/E2 region (Baker C. C. (1993) In: S. O'Brien (ed.)Genetic maps: locus maps of complex genomes, Cold Spring HarborLaboratory Press, p. 1.134-1.146). This integration leads to thederegulation of the expression of the viral E6 and E7 transforminggenes. This may be due in part to the release of the E6/E7 promoter fromthe repressor effects of E2. The data set forth below demonstrates thatthe E2 protein can inhibit growth of HPV-immortalized cells. Moreover,we have shown that HPV-positive cervical carcinoma cells are sensitiveto the reintroduction of HPV E2 proteins; their growth is inhibited byexpression of E2_(ad/db) proteins. While not being bound by anyparticular theory, the growth-suppressive effect of E2 is presumablymediated by transcriptional repression of E6 and E7 expression from theHPV-16 P₉₇ promoter or, e.g., in HeLA cells, from the HPV-18 p108promoter.

By virtue of the present invention, there is provided methods andcompositions for interfering with the proliferation of cells infectedand/or transformed by papillomaviruses. The processes and compositionsof this invention may be used to treat any mammal, including humans.According to this invention, mammals are treated by the pharmaceuticallyacceptable administration of an E2_(ad/db) protein, either directly orby gene transfer techniques, to reduce the symptoms of the specificpapillomavirus-associated disease, or to prevent their recurrence.Diseases which may be treated by the processes and compositions of thisinvention are those caused by the etiological agent, papillomavirus.Such diseases include, for example, epithelial malignancies, anogenitalmalignancies, such as cervical cancer, malignant lesions, benignlesions, papillomacarcinomas, papilloadenocystomas, papillomaneurophathicum, papillomatosis, cutaneous and mucosal papillomas,condylomas, oral, pharyngeal, laryngeal, and tongue papillomas,fibroblastic tumors and other pathological conditions associated withpapillomavirus. The E2-derived compositions of this invention may alsobe used to treat epithelial and internal fibropapillomas in animals.

A wide variety of warts are found on human skin and are caused by thehuman papilloma virus (HPV). For example, the following types of wartsare found on human skin and are caused by the human papilloma virus(HPV): common warts (verruca vulgaris), plantar warts, palmar warts,planar warts (verruca plana), mosaic warts, and venereal warts(condyloma accuminatum). These skin growths are unsightly, irritating,and potentially oncogenic (carcinogenic), and their removal is desired.

Genital warts, also referred to as venereal warts and condylomataacuminata, are one of the most serious manifestations of HPV infection.As reported by the Center for Disease Control, the sexual mode oftransmission of genital warts is well established and the incidence ofgenital warts is on the increase. The seriousness of genital warts isunderlined by the finding that HPV DNA can be found in all grades ofcervical intraepithelial neoplasia (CIN I-III) and that a specificsubset of HPV types can be found in carcinoma in situ of the cervix.Consequently, women with genital warts, containing specific HPV typesare now considered at high risk for the development of cervical cancer.Current treatments for genital warts are inadequate. According to thepresent invention, a method of treating a patient having one or moregenital warts comprises the administration of a pharmaceuticalcomposition including an E2_(ad/db) polypeptide, or a gene constructencoding the E2 protein, so as to inhibit growth of the wart. Inpreferred embodiments, the wart(s) are contacted directly with thepharmaceutical composition. The subject method can be used to treat,e.g, condyloma acuminata and/or flat cervical warts.

Laryngeal papillomas are benign epithelial tumors of the larynx. Two PVtypes, HPV-6 and HPV-11, are most commonly associated with laryngealpapillomas. According to the method of the present invention, laryngealpapillomas are treated administrating a pharmaceutical compositionincluding the therapeutic E2 polypeptide, or a gene construct encodingthe E2 polypeptide, so as to inhibit growth of the papillomas.

The most common disease associated with papillomavirus infection arebenign skin warts. Common warts generally contain HPV types 1, 2, 3, 4or 10. These warts typically occur on the soles of feet, plantar warts,or on the hands. Common skin warts are most often found in children andyoung adults. Later in life the incidence of common warts decreasespresumably due to immunologic and physiologic changes. Plantar warts canoften be debilitating and require surgical removal and they frequentlyreoccur after surgery. As above, patients suffering from common wartscan be treated by the administration of a effective amount of an E2protein according to the present invention, or a gene therapy constructwhich encodes the therapeutic E2 protein. In preferred embodiments, theprotein or gene construct are applied, in the appropriate formulations,directly to the area of the skin afflicted with the wart(s). Similarmethods and compositions may be useful in the treatment ifepidermodysplasia verruciformis (EV), a rare genetically transmitteddisease which is characterized by disseminated flat warts that appear assmall reddish macules.

In addition, the subject method and compositions may be used to treatlesions resulting from cellular transformation for which HPV is aetiological agent, e.g., in the treatment of cervical cancer or, e.g.,in the treatment of metastasized HPV positive tumors.

Accordingly, the invention has several advantages. In one embodiment,the invention may be used in the treatment of a patient infected withmultiple papillomavirus subtypes without activating viral replication.Moreover, the E2 protein of one human papillomavirus subtype mayfunctionally substitute for the E2 protein of a different viral subtypein the regulation of both viral transcription and replication(DelVecchio et al., (1992) Am. J. Virol. 66:5949-5958; Chiang et al.,PNAS 89:5799-803, 1992). Thus, providing forms of E2 selected tomaintain growth suppression without activating viral replication in, forexample, an HPV positive lesion, e.g., a cervical carcinoma, can beexpected to provide cross-protective activity in other HPV positivecells in the patient harboring entirely different viral subtypes (Donget al., (1994) J. Virol. 68:1115-1127). The E2 residue E39 has beenshown to be indispensable for the viral DNA replication function of allpapillomaviruses for which it has been tested. Thus, the E39A mutationand the altered hinge mutations exemplified herein are idealtherapeutics for suppressing the growth of HPV positive cells, e.g.,tumor cells or cells associated with a mixed papillomavirus infection.For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

As used herein, the term “E2 gene” or “recombinant E2 gene” refers to anucleic acid comprising an open reading frame encoding a papillomavirusE2 polypeptide.

As used herein, the term “E2_(ad/db) polypeptide” is intended to includeany papillomavirus E2 polypeptide that comprises a minimaltransactivation domain (i.e., activation domain or “ad”) and a minimalDNA binding domain (i.e., “db”) and is capable of repressing E6/E7expression or inhibiting cell growth in papillomavirus-infected cells.Accordingly, the “E2_(ad/db) ” may be derived from any species, e.g.,bovine, human, or rabbit and any papillomavirus subtype. E2_(ad/db)polypeptides also include modified E2_(ad/db) polypeptides which, e.g.,may have one or more alterations, e.g., amino acid substitutions (e.g.,E39A), deletions, e.g., a deletion of the hinge region (e.g., BPVE2₂₂₀₋₃₀₉), or additions such that the polypeptide selectivelysuppresses cell growth in papillomavirus-infected cells in a mannersuperior to a corresponding unaltered wild type E2 polypeptide.Corresponding alterations may be made in other E2 polypeptides ofdifferent subtypes in order to achieve the same desired activity.Preferred E2_(ad/db) polypeptides are selected for their inability topromote papillomavirus replication. In addition, preferred E2_(ad/db)polypeptides are selected for there inability to repress growth in cellsinfected by like and non-like viral subtypes.

As used herein, the term “lacking the ability to promote papillomavirusreplication” is intended to include any reduction or elimination in theability of an E2_(ad/db) polypeptide to enhance or promotepapillomavirus replication as compared to the activity found in acorresponding wild type E2 polypeptide.

As used herein, the term “reduced cell growth” is intended to includeany reduction in cell growth or, e.g., the complete cessation of cellgrowth causing, e.g., apoptosis, in one or more papillomavirus-infectedcells when treated with an E2_(ad/db) polypeptide or gene constructencoding such a polypeptide. Reductions in cell growth may be measured,e.g., using the colony assay as described herein.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of one of an E2 gene is under thecontrol of a promoter sequence (or other transcriptional regulatorysequence) which controls the expression of the recombinant gene in acell-type in which expression is intended. It will also be understoodthat the recombinant gene can be under the control of transcriptionalregulatory sequences which are the same or which are different fromthose sequences which control transcription of the naturally-occurringforms of E2 proteins.

The term “gene construct”, with respect to the subject E2 proteins,refers to a vector, plasmid, viral genome or the like which includes anE2 coding sequence, can transfect cells, preferably mammalian cells, andcan cause expression of the E2 coding sequence in cells transfected withthe construct. The term “gene construct” does not include a wild-typepapillomavirus genome, and preferably does not include expressiblecoding sequences for one or more of a papillomavirus E6 or E7 proteins.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which effects expressionof the selected DNA sequence in specific cells of a tissue, such ascells of hepatic or pancreatic origin, e.g. neuronal cells. The termalso covers so-called “leaky” promoters, which regulate expression of aselected DNA primarily in one tissue, but cause expression in othertissues as well.

As used herein, the term “pharmaceutically acceptable” refers to acarrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredients and which is not toxic tothe hosts to which it is administered. The administration(s) may takeplace by any suitable technique, including subcutaneous and parenteraladministration, preferably parenteral. Examples of parenteraladministration include intravenous, intraarterial, intramuscular, andintraperitoneal, with intravenous being preferred.

As used herein, the term “prophylactic or therapeutic” treatment refersto administration to the host of the papillomavirus medicament. If it isadministered prior to exposure to the virus, the treatment isprophylactic (i.e., it protects the host against infection), whereas ifadministered after infection or initiation of the disease, the treatmentis therapeutic (i.e., it combats the existing infection or cancer).

As used herein the term “papillomavirus disease” refers to any kind ofdisease caused by the virus, including cancers and warts.

The term “cell-proliferative disorder” denotes malignant as well asnonmalignant cell populations which morphologically often appear todiffer from the surrounding tissue.

Operably linked is intended to mean that the nucleotide sequence islinked to a regulatory sequence in a manner which allows expression ofthe nucleotide sequence. Regulatory sequences are art-recognized and areselected to direct expression of the subject papillomavirus E2 proteins.Accordingly, the term transcriptional regulatory sequence includespromoters, enhancers and other expression control elements. Suchregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences, sequences that control the expression of a DNA sequence whenoperatively linked to it, may be used in these vectors to express DNAsequences encoding papillomavirus E2 polypeptides of this invention.Such useful expression control sequences, include, for example, a viralLTR, such as the LTR of the Moloney murine leukemia virus, the early andlate promoters of SV40, adenovirus or cytomegalovirus immediate earlypromoter, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage, the control regions for fd coatprotein, the promoter for 3-phosphoglycerate kinase or other glycolyticenzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast a-mating factors, the polyhedron promoter of the baculovirussystem and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof. It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other proteins encoded by thevector, such as antibiotic markers, should also be considered. In oneembodiment, the expression vector includes a recombinant gene encoding apeptide having an agonistic activity of a subject E2 polypeptide, oralternatively, encoding a peptide which is an antagonistic form of theE2 protein. Such expression vectors can be used to transfect cells andthereby produce polypeptides, including fusion proteins, encoded bynucleic acids as described herein.

I. Therapeutic E2 Proteins

The E2_(ad/db) proteins utilized therapeutically by the presentinvention include both an activation domain (ad) and a DNAbinding/dimerization domain (db). As described in the appended examples,the E2 transactivation and DNA binding/dimerization domains were eachnecessary for E2-mediated growth suppression of PV-infected andPV-transformed cells. E2 mutants which lacked a hinge region, e.g., butwhich retained the transactivation and DNA binding/dimerization domains,while defective in replication function nevertheless retained theability to suppress growth of PV-transformed cells, indicating that thegrowth-suppressive properties of E2 can be unlinked from its DNAreplication properties. As presented in the examples, an E2_(ad/db)protein, i.e., E2 (E39A), having only a single lesion in thetransactivation domain also had these desired characteristics.

The general structure of the papillomavirus E2 protein is well known.See, for example, McBride et al. (1991) J. Biol Chem 266:18411-18414;and Giri et al. (1988) EMBO J 7:2823-2829. Both the activation domainand the DNA binding domain of a papillomavirus E2 protein will bereadily identified by one of ordinary skill in the art. For instance, aregion of approximately 200 amino acids at the N terminus of E2corresponds to the activation domain (FIG. 1). This domain contains tworegions predicted to form acidic amphipathic helices (e.g., using thealgorithms developed by Chou and Fasman), which have been shown to beimportant for the activation function of E2. DNA binding function isprovided by the C-terminal 85 amino acids or so of the E2 protein (FIG.1). Dimerization is also mediated by the DNA binding domain. Theactivation and DNA binding domains are recognized in humanpapillomavirus E2 proteins, including E2 proteins from the high riskviruses, e.g., HPV-16, -18, -31 or -33.

According to the present invention, the therapeutic E2 protein of thesubject method includes both the activation and DNA binding domains. Toillustrate, an exemplary E2_(ad/db) protein suitable for use in thepresent method is generated with a human papillomavirus E2 protein, andpreferably from a high risk HPV. For instance, therapeutic compositionsof the present invention can be derived from an HPV-16 E2 protein andincludes an activation domain corresponding to approximately Met1-Ser198of SEQ ID No. 2 and a DNA binding domain corresponding to approximatelyCys281-Ile356 of SEQ ID No. 2. Likewise, the subject method can employan E2 protein which includes an HPV-18 E2 activation domaincorresponding to approximately Met1-Asn203 and a DNA binding domaincorresponding to approximately Cys282-Met365 of SEQ ID No. 4.

In a particular embodiment, the invention provides a therapeutic in theform of a modified E2 protein having an glutamic acid (E) to alanine (A)amino acid substitution at residue position 39 (e.g., HPV16 E2 (E39A)).Moreover, as this residue is conserved among many E2 proteins found invarious bovine and human papillomavirus strains (e.g., BPV1, HPV6b,HPV11, HPV18, HPV31, HPV1A, and HPV57), including several high riskstrains, (e.g., HPV16 and HPV18), the present invention also encompassesany corresponding mutation in any of these E2 polypeptides from anyspecies (for a review of various species strains of papillomaviruses,see, e.g., Fields et al. (1996) Fields Virology). Further, such amutation need not be an alanine substitution, but may also be anysimilar substitution that results in an E2 polypeptide capable ofinhibiting cell growth but not capable of promoting papillomavirusreplication. Accordingly, the invention encompasses a modified oraltered E2 polypeptide, and methods for identifying such a polypeptide,that can repress cell growth, e.g., in papillomavirus-infected cells,but does not promote papillomavirus replication. The invention alsoencompasses assays for identifying E2 polypeptides having thesecharacteristics as described herein.

The examples set forth below illustrate that the hinge region (FIG. 1),which joins the E2 polypeptide sequence between the activation and DNAbinding domains, is not necessary for the growth suppressive activity ofthe papillomavirus E2 protein. While in preferred embodiments an intactE2 protein is utilized in the present method, e.g., a contiguouspolypeptide corresponding to the activation domain, hinge region and DNAbinding domain, the present invention also contemplates the use of E2proteins which lack all or a portion of the hinge region. For example, arecombinant E2 protein can be provided in which the hinge region isdeleted, the activation and DNA binding domains being directlycontiguous with one and other. The examples provide the illustrativeE2₆₆ ₂₂₀₋₃₀₉ protein to demonstrate the growth inhibitory activity ofsuch constructs. Methods of making fusion proteins (or deletions as thecase may be) are well known in the art. Essentially, the joining ofvarious DNA fragments coding for different domains is performed inaccordance with conventional techniques, employing blunt-ended orstagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the E2_(ad/db)gene can be synthesized by conventional techniques including automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed to generate the E2_(ad/db) gene sequence (see, for example,Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley &Sons: 1992).

In some instances where the hinge region has been deleted it may bedesirable to introduce an unstructured polypeptide linker region betweenthe DNA binding domain and the activation domain in place of thenaturally occurring hinge region. This linker can facilitate enhancedflexibility of the protein allowing the two domains to freely and(optionally) simultaneously interact with a DNA and cellular proteins byreducing steric hindrance between the two domains, as well as allowingappropriate folding of each portion to occur. The linker can be ofnatural origin, such as a sequence determined to exist in random coilbetween two domains of a protein. Alternatively, the linker can be ofsynthetic origin. For instance, the sequence (Gly₄Ser)₃ can be used as asynthetic unstructured linker. Linkers of this type are described inHuston et al. (1988) PNAS 85:4879; and U.S. Pat. Nos. 5,091,513 and5,258,498, and can be readily incorporated by recombinant techniques.

In still other embodiments, the activation and DNA binding domains canbe provided in the same molecule by chemical cross-linking. For example,there are a large number of chemical cross-linking agents that are knownto those skilled in the art and useful for cross-linking the activationand DNA binding domains of an E2 protein to provide a single molecule.In an exemplary embodiment, the cross-linking agents areheterobifunctional cross-linkers which can be used to link theactivation and DNA binding domains in a stepwise manner.Heterobifunctional cross-linkers provide the ability to design morespecific coupling methods for conjugating proteins, thereby reducing theoccurrences of unwanted side reactions such as homo-protein polymers. Awide variety of heterobifunctional cross-linkers are known in the art.These include: succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),m-Maleimidobenzoyl-N-hydroxy-succinimide ester (MBS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl4-(p-maleimido-phenyl)butyrate (SMPB),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC);4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-tolune (SMPT),N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl6-[3-(2-pyridyldithio) propionate]hexanoate (LC-SPDP). Thosecross-linking agents having N-hydroxysuccinimide moieties can beobtained as the N-hydroxy-sulfosuccinimide analogs, which generally havegreater water solubility. In addition, those cross-linking agents havingdisulfide bridges within the linking chain can be synthesized instead asthe alkyl derivatives so as to reduce the amount of linker cleavage invivo.

In addition to the heterobifunctional cross-linkers, there exists anumber of other cross-linking agents including homobifunctional andphotoreactive cross-linkers. Disuccinimidyl suberate (DSS),bismaleimidohexane (BMH) and dimethylpimelimidate.2 HCl (DMP) areexamples of useful homobifunctional cross-linking agents, andbis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED) andN-succinimidyl-6(4′-azido-2′-nitrophenyl-amino)hexanoate (SANPAH) areexamples of useful photoreactive cross-linkers for use in generating thesubject E2_(ad/db) proteins. For a recent review of protein couplingtechniques, see Means et al. (1990) Bioconjugate Chemistry 1:2-12,incorporated by reference herein.

One particularly useful class of heterobifunctional cross-linkers,included in the representative lists above, contain the primary aminereactive group, N-hydroxysuccinimide (NHS), or its water soluble analogN-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine epsilongroups) at alkaline pH's are unprotonated and react by nucleophilicattack on NHS or sulfo-NHS esters. This reaction results in theformation of an amide bond, and release of NHS or sulfo-NHS as aby-product. Another reactive group useful as part of aheterobifunctional cross-linker is a thiol reactive group. Common thiolreactive groups include maleimides, halogens, and pyridyl disulfides.Maleimides react specifically with free sulfhydryls (cysteine residues)in minutes, under slightly acidic to neutral (pH 6.5-7.5) conditions.Halogens (iodoacetyl functions) react with —SH groups at physiologicalpH's. Both of these reactive groups result in the formation of stablethioether bonds.

Preparing conjugates of the E2 protein domains using heterobifunctionalreagents will typically be a two-step process involving the aminereaction and the sulfhydryl reaction. For the first step, the aminereaction, the E2 polypeptide sequence should contain a primary amine.This can be lysine epsilon amines or an unprotected primary α-aminefound at the N-terminus. The polypeptide should not contain freesulfhydryl groups. The polypeptide chain can be modified so that allsulfhydryls are blocked using for instance, N-ethylmaleimide (see Partiset al. (1983) J. Pro. Chem. 2:263; and Riddles et al. (1979) Anal.Biochem. 94:75).

The reaction buffer should be free of extraneous amines and sulfhydryls.The pH of the reaction buffer should be 7.0-7.5. This pH range preventsmaleimide groups from reacting with amines, preserving the maleimidegroup for the second reaction with sulfhydryls. The NHS-ester containingcross-linkers have limited water solubility. They should be dissolved ina minimal amount of organic solvent (DMF or DMSO) before introducing thecross-linker into the reaction mixture. The cross-linker/solvent formsan emulsion which will allow the reaction to occur.

The sulfo-NHS ester analogs are more water soluble, and can be addeddirectly to the reaction buffer. Buffers of high ionic strength shouldbe avoided, as they have a tendency to “salt out” the sulfo-NHS esters.To avoid loss of reactivity due to hydrolysis, the cross-linker is addedto the reaction mixture immediately after dissolving the proteinsolution.

Once the reaction is completed, the first polypeptide portion of the E2protein is now activated with a sulfhydryl reactive moiety. Theactivated protein may be isolated from the reaction mixture by simplegel filtration or dialysis. To carry out the second step of thecross-linking, the sulfhydryl reaction, the second E2 polypeptideportion must contain a free sulfhydryl, e.g., an unprotected cysteineresidue.

Maleimides react specifically with -SH groups at slightly acidic toneutral pH ranges (6.5-7.5). A neutral pH is sufficient for reactionsinvolving halogens and pyridyl disulfides. Under these conditions,maleimides generally react with —SH groups within a matter of minutes.Longer reaction times are required for halogens and pyridyl disulfides.The first sulfhydryl reactive-protein prepared in the amine reactionstep is mixed with the second E2 polypeptide fragment under theappropriate buffer conditions. The E2_(ad)-E2_(db) conjugates can beisolated from the reaction mixture by methods such as gel filtration orby dialysis.

Generally, where purified stocks of an E2 protein (or fragments thereof)are required, e.g., for formulation intended for direct administration,conventional recombinant techniques can be employed to express andpurify an E2 protein. The term “recombinant E2 protein” refers to an E2polypeptide of the present invention which is produced by recombinantDNA techniques, wherein generally DNA encoding an E2_(ad/db) polypeptide(or discrete portions thereof) is inserted into a suitable expressionvector which is in turn used to transform a host cell to produce theheterologous protein. Moreover, the phrase “corresponding to”, withrespect to a recombinant E2 gene, is meant to include within the meaningof “recombinant protein” those proteins having an amino acid sequence(s)of native E2 polypeptides, or an amino acid sequence similar theretowhich is generated by mutations including substitutions and deletions ofa naturally occurring form of an E2 protein.

For example, a host cell transfected with a nucleic acid vectordirecting expression of a nucleotide sequence encoding an E2 polypeptidecan be cultured under appropriate conditions to allow expression of thepolypeptide to occur. The E2 polypeptide may include a signal sequenceand be secreted and isolated from a mixture of cells and mediumcontaining the recombinant E2 polypeptide. Alternatively, thepolypeptide may be retained cytoplasmically, as it normally is, and thecells harvested, lysed and the protein isolated. The recombinant E2polypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and affinity purification.

Thus, a nucleotide sequence derived from the cloning of an E2 openreading frame (ORF), encoding all or a selected portion of thefull-length protein, can be used to produce a recombinant form of an E2polypeptide via microbial or eukaryotic cellular processes. Ligating thepolynucleotide sequence into a gene construct, such as an expressionvector, and transforming or transfecting into hosts, either eukaryotic(yeast, avian, insect or mammalian) or prokaryotic (bacterial cells),are standard procedures used in producing other well-known PV proteins,e.g. E6, E7 and the like. Similar procedures, or modifications thereof,can be employed to prepare recombinant E2 polypeptides by microbialmeans or tissue-culture technology in accord with the compositions ofthe subject invention.

Moreover, it is widely appreciated that fusion proteins can facilitatethe expression of proteins, and accordingly, can be used in theexpression of the an E2_(ad/db) polypeptides of the present invention.For example, E2_(ad/db) polypeptides can be generated asglutathione-S-transferase (GST-fusion) proteins. Such GST-fusionproteins can enable easy purification of the E2_(ad/db) polypeptide, asfor example by the use of glutathione-derivatized matrices (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.(N.Y.: John Wiley & Sons, 1991)). In another embodiment, a fusion genecoding for a purification leader sequence, such as apoly-(His)/enterokinase cleavage site sequence, can be used at theN-terminus of the E2_(ad/db) protein in order to permit purification ofthe poly(His)-E2_(ad/db) protein by affinity chromatography using a Ni²⁺metal resin. The purification leader sequence can then be subsequentlyremoved by treatment with enterokinase (e.g., see Hochuli et al. (1987)J. Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

In the instance of direct delivery of a therapeutic protein (describedinfra), some uptake mechanisms for the protein may involve passagethrough lysosomes. As long half-life in the target cells is desirable,an E2 protein of this invention may be modified to increase itsresistance to protease degradations and/or acid hydrolysis and, in turn,increase the half-life of the polypeptide in circulation and cells. Inone embodiment of the present invention, the protease resistance of anE2 protein is increased by incorporation of D-amino acids instead ofL-amino acids at some or all residues of the polypeptide, e.g., as aretro-enantio or retro-inverso peptide. In another embodiment, thepeptide backbone is modified using other amide mimetics, such astrans-olefins (Shue et al. (1987) Tetrahedron Letters 28:3225) orphosphate derivatives (Loots et al. in Peptides: Chemistry and Biology,(Escom Science Publishers, Leiden, 1988, p. 118)) in order to protectthe E2 protein from degradation. In still another embodiment, the aminoterminus, or carboxy terminus, or both termini of an E2 polypeptide areblocked by chemical modification. In a further embodiment of thisinvention, lysosomal proteases are inhibited by an E2 protein in acomposition comprising a lysomotrophic agent, such as chloroquine,amantadine, monensin, methylamine, or ammonium chloride.

II. Gene Therapy

In one aspect, the present invention relates to gene therapy constructscontaining a nucleic acid encoding a papillomavirus E2_(ad/db)polypeptide, preferably an HPV E2 protein, operably linked to at leastone transcriptional regulatory sequence. The gene constructs of thepresent invention are formulated to be used as a part of a gene therapyprotocol to deliver the subject therapeutic protein to apapillomavirus-infected or -transformed cell in an animal.

Any of the methods known to the art for the insertion of DNA fragmentsinto a vector may be used to construct expression vectors consisting ofappropriate transcriptional/translational control signals and thedesired E2-encoding nucleotide sequence. See, for example, Maniatis T.,Fritsch E. F., and Sambrook J. (1989): Molecular Cloning (A LaboratoryManual), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; andAusubel F. M., Brent R., Kingston R. E., Moore, D. D., Seidman J. G.,Smith J. A., and Struhl K. (1992): Current Protocols in MolecularBiology, John Wiley & Sons, New York. These methods may include in vitroDNA recombinant and synthetic techniques and in vivo geneticrecombination. Expression of a nucleic acid sequence encoding anE2_(ad/db) protein may be regulated by a second nucleic acid sequence sothat the protein is expressed in a host infected or transfected with therecombinant DNA molecule. For example, expression of E2 may becontrolled by any promoter/enhancer element known in the art. Thepromoter activation may be tissue specific or inducible by a metabolicproduct or administered substance.

Promoters/enhancers which may be used to control the expression of theE2 gene in vivo include, but are not limited to, the native E2 promoter,the cytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J.Exp. Med., 169:13), the human β-actin promoter (Gunning et al. (1987)PNAS 84:4831-4835), the glucocorticoid-inducible promoter present in themouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig etal. (1984) Mol. Cell Biol. 4:1354-1362), the long terminal repeatsequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al.(1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), the SV40 early or late region promoter (Bernoist et al.(1981) Nature 290:304-310; Templeton et al. (1984) Mol. Cell Biol.,4:817; and Sprague et al. (1983) J. Virol., 45:773), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (RSV)(Yamamoto et al., 1980, Cell, 22:787-797), the herpes simplex virus(HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS82:3567-71), and the herpes simplex virus LAT promoter (Wolfe et al.(1992) Nature Genetics, 1:379-384).

Expression constructs of the subject E2 polypeptides may be administeredin any biologically effective carrier, e.g. any formulation orcomposition capable of effectively delivering the recombinant gene tocells in vivo. Approaches include insertion of the E2 gene in viralvectors including recombinant retroviruses, adenovirus, adeno-associatedvirus, and herpes simplex virus-1, or recombinant eukaryotic plasmids.Viral vectors transfect cells directly; plasmid DNA can be deliveredwith the help of, for example, cationic liposomes (lipofectin) orderivatized (e.g antibody conjugated), polylysine conjugates, gramacidinS, artificial viral envelopes or other such intracellular carriers, aswell as direct injection of the gene construct or CaPO₄ precipitationcarried out in vivo. It will be appreciated that because transduction ofappropriate target cells represents the critical first step in genetherapy, choice of the particular gene delivery system will depend onsuch factors as the phenotype of the intended target and the route ofadministration, e.g. locally or systemically.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid encoding theparticular E2 polypeptide desired. Infection of cells with a viralvector has the advantage that a large proportion of the targeted cellscan receive the nucleic acid. Additionally, molecules encoded within theviral vector, e.g., the recombinant E2 protein, are expressedefficiently in cells which have taken up viral vector nucleic acid.

One viral gene delivery system useful in the present invention utilizesadenovirus-derived vectors for in vivo expression of an E2 protein. Thegenome of an adenovirus can be manipulated such that it encodes andexpresses a gene product of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See for exampleBerkner et al. (1988) Biotechniques 6:616; Rosenfeld et al. (1991)Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.Adenoviruses (Ads) are a relatively well characterized, homogeneousgroup of viruses. Roughly 100 different adenoviruses, including nearly50 serotypes isolated from humans, have been identified to date.

Suitable adenoviral vectors derived from the adenovirus strain Ad type 5dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are wellknown to those skilled in the art. Recombinant adenoviruses can beadvantageous in certain circumstances in that they can be used to infecta wide variety of cell types, including epithelial cells (see, forexample, Goldman et al. (1995) J Virol 69:5951-8; Clayman et al. (1985)Cancer Gene Therapy 2:105-111; and Rosenfeld et al. (1992), supra),including genitourinary epithelia (Bass et al. (1995) Cancer GeneTherapy 2:97-104). Furthermore, the virus particle is relatively stableand amenable to purification and concentration, and as above, can bemodified so as to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267).

Most replication-defective adenoviral vectors currently in use andtherefore favored by the present invention are deleted for all or partsof the viral E1 and E3 genes but retain as much as 80% of the adenoviralgenetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner etal., supra; and Graham et al. in Methods in Molecular Biology, E. J.Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127).Expression of the inserted papillomavirus E2 gene can be under controlof, for example, the E1A promoter, the major late promoter (MLP) andassociated leader sequences, the E3 promoter, or exogenously addedpromoter sequences.

Ad vectors of the present invention can be constructed by ligation of anE2_(ad/db) coding sequence with adenoviral sequences contained inbacterial plasmids. See, for example, Berkner et al. (1983) NucleicAcids Res. 11: 6003-6020; Haj-Ahmad et al. (1986) J. Virol. 57: 267-274;and Stow (1981) J. Virol. 37: 171-180. In a preferred strategy, twoplasmids which together contain sequences comprising the entire Adgenome and the E2_(ad/db) sequence are recombined. A number ofconditionally defective plasmid systems have been developed making theconstruction of vectors simpler and reducing the number of subsequentanalyses required to identify recombinant viruses. McGrory et al. (1988)Virol. 163: 614-617; Ghosh-Choudhury et al. (1986) Gene 50: 161-171; andMittal et al. (1993) Virus Res. 28: 67-90. The Graham et al. PCTpublication WO 95/00655, and corresponding Bett et al. (1994) PNAS91:8802-8806 publication, describe a state-of-the-art set of vectorswhich are useful in generating adenovirus-based vectors and areparticularly attractive to generating the E2_(ad/db) expressionconstructs of the present invention.

The following properties are desirable in the design of an adenovirusvector to transfer the gene for E2 to papillomavirus-transformed cellsof a patient. The vector should allow sufficient expression of the E2protein, while producing minimal viral gene expression. There should beminimal viral DNA replication and ideally no virus replication. Finally,recombination to produce new viral sequences and complementation toallow growth of the defective virus in the patient should be minimized.An exemplary adenovirus vector encoding E2 (Ad5/E2) is described below.

FIG. 2 is a map of an illustrative Ad5/E2 construct. This vectorincludes viral DNA derived from the common relatively benign adenovirus5 serotype. The E1a and E1b regions of the viral genome, which areinvolved in early stages of viral replication have been deleted, as hasthe E3 region (though this is optional). The therapeutic papillomavirusE2 coding sequence is inserted into the viral genome in place of theE1a/E1b region (ΔE1) and transcription of the E2 sequence is driven bythe human immediate early promoter region (pCMV) of the humancytomegalovirus.

Merely for illustration, the E2 ORF of a papillomavirus, such as an HPV,are amplified with primers which add HindIII and XbaI restriction sitesto the 5′ and 3′ ends of the ORF, respectively. For example, asdescribed by Del Vecchio et al. (1992) J Virology 66:5949-5958, PCR canbe used to isolate HPV-16 nucleotides 2756 to 3855 (HPV-16 E2) ornucleotides 2817 to 3999 (HPV-18 E2). The amplified E2 coding sequencecan then be cloned into the HindIII/XbaI site of the pCDM8 plasmid (InVitrogen catalog V308-20) to provide the E2 coding sequence downstreamof pCMV. The E2 coding sequence is also flanked at its 3′ end by SV40sequences (SV40intron/pA) which add the transcription termination andpolyadenylation signals to an E2 transcript.

The resulting plasmid is linearized with SacII, and the sequencecorresponding to the CMV promoter, E2 ORF and SV40intron/pA portion ofthe plasmid is amplified using primers which add a HindIII site at the5′ end of the CMV promoter and preserve the BamHI site at the 3′ end ofthe SV40 sequences. The resulting PCR product, designatedpCMV-E2_(ad/db)-SV40intron/pA, is cleaved by limited digestion withHindIII and BamHI and the appropriate fragment isolated, e.g. whichincludes the pCMV, E2 ORF and SV40 sequences.

The resulting HindIII/BamHI fragment is subsequently cloned into thecorresponding restriction sites of the pΔE1sp1B vector (see Bett et al.(1994) PNAS 91:8802-8806). Following the protocols of Bett et al., theresulting shuttle vector is cotransfected into 293 cells along with, forexample, the pHGG10 vector (see Bett et al., supra), and infectiousparticles isolated from the cell culture.

Adenoviral vectors currently in use retain most (≧80%) of the parentalviral genetic material. Recently, second-generation vector systemscontaining minimal adenoviral regulatory, packaging and replicationsequences have therefore been developed and may be used to deliver thetherapeutic E2 protein. In one embodiment, the E2 protein is expressedby a pseudo-adenovirus vector (PAV). PAVs contain adenovirus invertedterminal repeats and the minimal adenovirus 5′ sequences required forhelper virus dependent replication and packaging of the vector. Thesevectors contain no potentially harmful viral genes, and may be producedin reasonably high titers and maintain the tropism of the parent virusfor dividing and non-dividing human target cell types.

The PAV vector can be maintained as either a plasmid-borne construct oras an infectious viral particle. As a plasmid construct, PAV is composedof the minimal sequences from the wild type adenovirus necessary forefficient replication and packaging of these sequences and any desiredadditional exogenous genetic material, by either a wild-type ordefective helper virus.

In one embodiment, an exemplary PAV contains adenovirus 2 (Ad2)sequences including nucleotides (nt) 0-356 of Ad2 forming the 5′ end ofthe vector and the last 109 nt of Ad2 forming the 3′ end of theconstruct. These sequences include the Ad2 flanking inverted terminalrepeats and the 5′ ITR adjoining sequences containing the knownpackaging signal and E1a enhancer. PCT publication WO94/12649 describesvarious PAVs in which convenient restriction sites have beenincorporated into the fragments, allowing the insertion of E2-encodingsequences which can be packaged in the PAV virion and used for genetransfer (e.g. for gene therapy). The construction and propagation ofPAV is described in detail in WO94/12649. By not containing most nativeadenoviral DNA, the PAVs are less likely to produce a patient immuneresponse or to replicate in a host.

In addition, the PAV vectors can accommodate foreign DNA up to a maximumlength of nearly 36 kb. The PAV vectors therefore, while especiallyuseful for cloning larger genes can be used to transfer more than onegene, or more than one copy of the E2_(ad/db) gene. For example, PAVscan be used to deliver the therapeutic E2 gene in conjunction with othergenes such as p21^(CIP1) or p27^(KIP1).

Yet another viral vector system useful for delivery of a papillomavirusE2 gene is the adeno-associated virus (AAV). Adeno-associated virus is anaturally occurring defective virus that requires another virus, such asan adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). One advantageous feature of AAV derivesfrom its reduced efficacy for transducing primary cells relative totransformed cells (Halbert et al. (1995) J Virol 69:1473). Thus, thespectrum of infectivity for a recombinant E2-derived AAV will besomewhat restricted to papillomavirus transformed cells rather thansurrounding normal tissue.

Vectors containing as little as 300 base pairs of AAV can be packagedand can integrate. Space for exogenous DNA is limited to about 4.5 kb.An AAV vector such as that described in Tratschin et al. (1985) Mol.Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. Avariety of nucleic acids have been introduced into different cell typesusing AAV vectors (see for example Hermonat et al. (1984) Proc. Natl.Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol.4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39;Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993)J. Biol. Chem. 268:3781-3790).

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding anE2 protein rendering the retrovirus replication defective. Thereplication defective retrovirus is then packaged into virions which canbe used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines for preparing bothecotropic and amphotropic retroviral systems include ψCrip, ψCre, ψ2 andψAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including epithelial cells (see for example,Shillitoe et al. (1994) Cancer Gene Ther 1:193-204; Noel et al. (1994)Pediatr Gastroenterol Nutr 19:43-9; Archer et al. (1995) PNAS 91:6840-4;Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988)Proc. Natl. Acad Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl.Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad.Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (e.g. lactose to convertthe env protein to an asialoglycoprotein), as well as by generatingfusion proteins (e.g. single-chain antibody/env fusion proteins). Thistechnique, while useful to limit or otherwise direct the infection tocertain tissue types, can also be used to convert an ecotropic vector into an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the E2 gene of the retroviral vector.

Numerous retroviral gene delivery vehicles may be utilized within thecontext of the present invention, including for example EP 0,415,731; WO90/07936; WO 91/0285; WO 9403622; WO 9325698; WO 9325234; U.S. Pat. No.5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res.53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram etal., Cancer Res. 53:83-88, 1993; Takamiya et al., J. Neurosci. Res.33:493-503, 1992; Baba et al., J. Neurosurg. 79:729-735, 1993 (U.S. Pat.No. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91/02805). For example,retroviral gene delivery vehicles of the present invention may bereadily construction from a wide variety of retroviruses including forexample, B, C, and D type retroviruses as well as spumaviruses andlentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). Briefly, viruses are often classified according totheir morphology as seen under electron microscopy. Type “B”retroviruses appear to have an eccentric core, while type “C”retroviruses have a central core. Type “D” retroviruses have amorphology intermediate between type B and type C retroviruses. Suchretroviruses may be readily obtained from depositories or collectionssuch as the American Type Culture Collection (“ATCC” Rockville, Md.), orisolated from known sources using commonly available techniques.

Particularly preferred retroviruses for the preparation or constructionof retroviral gene delivery vehicles of the present invention includeretroviruses selected from the Mink-Cell Focus-Inducing Virus, MurineSarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma Virus.Particularly preferred Murine Leukemia Viruses include 4070A and 1504A(Hartley and Rowe (1976), J. Virol. 19:19-25), Abelson (ATCC No.VR-999), Friend, (ATCC No. VR-245), Graffi, Gross (ATCC No. VR-590),Kirsten, Harvey Sarcoma and Virus, Rauscher (ATCC NO. VR-998), andMoloney Murine Leukemia Virus (ATCC No. VR-190). Particularly preferredRous Sarcoma Viruses include Bratislava, Bryan high titer (e.g., ATCCNos. VR-334, VR-657, VR-726, VR-659, and VR-728), Bryan standard,Carr-Zilber, Engelbreth-Holm, Harris, Prague (e.g., ATCC Nos. VR-772,and 45033), and Schmidt-Ruppin (e.g. ATCC Nos. VR-724, VR-725, VR-354).

Any of the above retroviruses may be readily utilized in order toassemble or construct retroviral gene delivery vehicles given thedisclosure provided herein, and standard recombinant techniques (e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, 1989; Kunkle, PNAS 82:488, 1985). Inaddition, within certain embodiments of the invention, portions of theretroviral gene delivery vehicles may be derived from differentretroviruses. For example, within one embodiment of the invention,retroviral LTRs may be derived from a Murine Sarcoma Virus, at tRNAbinding site from a Rous Sarcoma Virus, a packaging signal from a MurineLeukemia Virus, and an origin of second strand synthesis from an AvianLeukosis Virus.

In some embodiments, retroviral vector constructs are providedcomprising a 5′ LTR, a tRNA binding site, a packaging signal, one ormore heterologous sequences including the E2-coding sequence, an originof second strand DNA synthesis and a 3′ LTR, but lacking gag/pol or envcoding sequences. Briefly, Long Terminal Repeats (“LTRs”) are subdividedinto three elements, designated U5, R and U3. These elements contain avariety of signals which are responsible for the biological activity ofa retrovirus, including for example, promoter and enhancer elementswhich are loaded within U3. LTRs may be readily identified in theprovirus due to their precise duplication at either end of the genome.

The tRNA binding site and origin of second strand DNA synthesis are alsoimportant for a retrovirus to be biologically active, and may be readilyidentified by one of skill in the art. For example, retroviral tRNAbinds to a tRNA binding site by Watson-Crick base pairing, and iscarried with retrovirus genome into a viral particle. The tRNA is thenutilized as a primer for DNA synthesis by reverse transcriptase. ThetRNA binding site may be readily identified based upon its location justdownstream from the 5′ LTR. Similarly, the origin of second strand DNAsynthesis is, as its name implies, important for the second strand DNAsynthesis of a retrovirus. This region, which is also referred to as thepoly purine tract, is located just upstream of the 3′LTR.

As an illustration, within one embodiment of the invention constructionof retroviral vector constructs which lack gag/pol or env sequences maybe accomplished by preparing retroviral vector constructs which lack anextended packaging signal. As utilized herein, the phrase “extendedpackaging signal” refers to a sequence of nucleotides beyond the minimumcore sequence which is required for packaging, that allows increasedviral titer due to enhanced packaging. As an example, for the MurineLeukemia Virus MoMLV, the minimum core packaging signal is encoded bythe sequence (counting from the 5′ LTR cap site) from approximatelynucleotide 144, up through the Pst I site (Nucleotide 567). See, forexample, the Jolly et al. PCT publication WO95/31566. The extendedpackaging signal of MoMLV includes the sequence beyond nucleotide 567 upthrough the start of the gag/pol gene (nucleotide 621), and beyondnucleotide 1040. Thus, within this embodiment retroviral vectorconstructs which lack extended packaging signal may be constructed fromthe MoMLV by deleting or truncating the packaging signal downstream ofnucleotide 567.

For example, the pCDM8 plasmid described above, e.g., containing thepCMV promoter, E2 ORF and SV40intron/pA, can be used to generate the PCRproduct pCMV-E2 ORF-SV40intron/pA, flanked by EcoRI restriction sites.The PCR product is digested with EcoRI, and the appropriate fragment,e.g., which includes the pCMV, E2 ORF and SV40 sequences, is isolatedand recloned into the MoMLV-derived pR2 vector (PCT publicationWO95/31566) which has been digested at its unique EcoRI site. ModifiedpR2 vectors with the E2 insert in the correct orientation are isolated.The retroviral vector is electroporated into a packaging cell line, andinfectious particles isolated from the cell culture.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a E2polypeptide in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the E2 gene by the targetedcell. Exemplary gene delivery systems of this type include liposomalderived systems, poly-lysine conjugates, and artificial viral envelopes.

In clinical settings, the gene delivery systems for the therapeutic E2gene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance, a pharmaceuticalpreparation of the gene delivery system can be introduced systemically,e.g by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470). In preferred embodiments, the gene therapyconstruct of the present invention is applied topically to HPV infectedor transformed cells of the skin or mucosal tissue. A papillomavirus E2gene construct can, in one embodiment, be delivered in a gene therapyconstruct by electroporation using techniques described, for example, byDev et al. ((1994) Cancer Treat Rev 20:105-115).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

III. Pharmaceutical Preparations of E2 Protein

According to another aspect of this invention, E2_(ad/db) proteins maybe administered directly to PV infected cells. Direct delivery of E2proteins may be facilitated by formulation of the protein in anypharmaceutically acceptable dosage form, e.g., for deliveryintratumorally, peritumorally, interlesionally, intravenously,intramuscularly, subcutaneously, periolesionally, or (preferably)topical routes, to exert local therapeutic effects.

Topical administration of the therapeutic is advantageous since itallows localized concentration at the site of administration withminimal systemic adsorption. This simplifies the delivery strategy ofthe agent to the disease site and reduces the extent of toxicologicalcharacterization. Furthermore, the amount of material to be applied isfar less than that required for other administration routes. Effectivedelivery requires the agent to diffuse into the affected cells.Successful intracellular delivery of agents not naturally taken up bycells has been achieved by exploiting the natural process ofintracellular membrane fusion, or by direct access of the cell's naturaltransport mechanisms which include endocytosis and pinocytosis (Duzgunes(1985) Subcellular Biochemistry 11:195-286). Such processes are alsouseful in the direct delivery and uptake of the subject E2_(ad/db)protein by papillomavirus-infected cells.

In one embodiment, the membrane barrier can be overcome by associatingthe E2 protein in complexes with lipid formulations closely resemblingthe lipid composition of natural cell membranes. In particular, thesubject E2_(ad/db) proteins are encapsulated in liposomes to formpharmaceutical preparations suitable for administration to living cellsand, in particular, suitable for topical administration to human skin.The Yarosh U.S. Pat. No. 5,190,762 demonstrates that proteins can bedelivered across the outer skin layer and into living cells, withoutreceptor binding, by liposome encapsulation.

These lipids are able to fuse with the cell membranes on contact, and inthe process, the associated E2 protein is delivered intracellularly.Lipid complexes can not only facilitate intracellular transfers byfusing with cell membranes but also by overcoming charge repulsionsbetween the cell membrane and the molecule to be inserted. The lipids ofthe formulations comprise an amphipathic lipid, such as thephospholipids of cell membranes, and form hollow lipid vesicles, orliposomes, in aqueous systems. This property can be used to entrap theE2 protein within the liposomes.

Liposomes offer several advantages: They are non-toxic and biodegradablein composition; they display long circulation half-lives; andrecognition molecules can be readily attached to their surface fortargeting to tissues. Finally, cost effective manufacture ofliposome-based pharmaceuticals, either in a liquid suspension orlyophilized product, has demonstrated the viability of this technologyas an acceptable drug delivery system.

Liposomes have been described in the art as in vivo delivery vehicles.The structure of various types of lipid aggregates varies, depending oncomposition and method of forming the aggregate. Such aggregates includeliposomes, unilamellar vesicles, multilameller vesicles, micelles andthe like, having particle sizes in the nanometer to micrometer range.Methods of making lipid aggregates are by now well-known in the art. Forexample, the liposomes may be made from natural and syntheticphospholipids, glycolipids, and other lipids and lipid congeners;cholesterol, cholesterol derivatives and other cholesterol congeners;charged species which impart a net charge to the membrane; reactivespecies which can react after liposome formation to link additionalmolecules to the liposome membrane; and other lipid soluble compoundswhich have chemical or biological activity.

In one embodiment, pH sensitive liposomes are a preferred type ofliposome for use with the present invention. One pathway for the entryof liposomes into cellular cytoplasm is by endocytosis into lysozymes oflow pH. Accordingly, liposomes which are stable at neutral pH butrelease their contents at acidic pH can be used to deliver enzymes intothe lysozymes of the cytoplasm, whereupon the contents are released.

Liposomes can be made sensitive to the low pH of the lysozymes by thelipid composition. In particular, pH sensitive liposomes can be preparedby using phospholipids which form lipid bilayers when charged but failto stack in an ordered fashion when neutralized. An example of such aphospholipid is phosphatidylethanolamine, which is negatively chargedabove pH 9. The net charge of a phospholipid can be maintained at a pHwhich would otherwise neutralize the head groups by including chargedmolecules in the lipid bilayer which themselves can become neutralized.Examples of these charged molecules are oleic acid and cholesterylhemisuccinate, which are negatively charged at neutral pH but becomeneutralized at pH 5. The effect of combining these together in a lipidbilayer is that at pH 9 all molecules are charged; at pH 7 the netnegative charge of the oleic acid and cholesteryl hemisuccinatemaintains the stability of the phosphatidylethanolamine, and at pH 5 allcomponents are protonated and the lipid membrane is destabilized.Additional neutral molecules, such as phosphatidylcholine, can be addedto the liposomes as long as they do not interfere with stabilization ofthe pH sensitive phospholipid by the charged molecules.

In another embodiment, the E2 polypeptide is formulated with apositively charged synthetic (cationic) lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),in the form of liposomes, or small vesicles, which can fuse with thenegatively charged lipids of the cell membranes of mammalian cells,resulting in uptake of the contents of the liposome (see, for example,Felgner et al. (1987) PNAS 84:7413-7417; and U.S. Pat. No. 4,897,355 toEppstein, D. et al.). Another cationic lipid which can be used togenerate E2-containing liposomes is the DOTMA analogue,1,2-bis(oleoyloxy)-3-(trimethyl-ammonio)propane (DOTAP) in combinationwith a phospholipid to form delivery vesicles.

Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) and/orLipofectAMINE™, commercially available reagents, can be used to deliverthe E2 protein directly into cells. Positively charged complexesprepared in this way spontaneously attach to negatively charged cellsurfaces, fuse with the plasma membrane, and can efficiently deliverfunctional E2 protein into, for example, keratinocytes. Sells et al.(1995) Biotechniques 19:72-76 describe a procedure for delivery ofpurified proteins into a variety of cells using such polycationic lipidpreparations.

A significant body of information is emerging regarding the use of othercationic lipids for the delivery of macromolecules into cells. Othersuitable lipid vesicles for direct delivery of the E2 protein includevesicles containing a quaternary ammonium surfactant (Ballas et al.(1988) Biochim. Biophys Acta 939:8-18); lipophilic derivatives ofspermine (Behr et al. (1989) PNAS 86:6982-6986).

The lipid formulations of the subject E2 protein can be used inpharmaceutical formulations to deliver the E2 protein by various routesand to various sites in the animal body to achieve the desiredtherapeutic effect. Local or systemic delivery of the therapeutic agentcan be achieved by administration comprising application or insertion ofthe formulation into body cavities, inhalation or insufflation of anaerosol, or by parenteral introduction, comprising intramuscular,intravenous, intradermal, peritoneal, subcutaneous and topicaladministration.

Topical formulations are those advantageously applied to the skin ormucosa. Target mucosa can be that of the gastrointestinal tract,comprising the mouth, larynx, esophagus and stomach, or the vaginal,vulvar, penal or anorectal mucosa. Other target tissues can be theaccessible epidermal tissues which are infected by HPV. Lipids presentin topical formulations can act to facilitate introduction oftherapeutic E2 protein into the target tissue, such as the stratum orcorneum of the skin, by perturbing the barrier properties of theprotective membrane, or by introducing perturbing agents or penetrationenhancers such as Azone™ or by promoting the activity of thesepenetration enhancers.

Other pharmaceutical formulations comprising the cationic lipids of theinvention are topical preparations containing an anesthetic orcytostatic agent, immunomodulators, bioactive peptides oroligonucleotides, sunscreens or cosmetics. Preparations for topical useare conveniently prepared with hydrophilic and hydrophobic bases in theform of creams, lotions, ointments or gels; alternatively, thepreparation may be in the form of a liquid that is sprayed on the skin.The effect of the cationic lipids is to facilitate the penetration ofthe active antiviral agent through the stratum corneum of the dermis.

The composition and form of pharmaceutical preparations comprising theliposome, in combination with the E2 protein, can vary according to theintended route of administration.

Also, by suitable modifications of the liposome membranes, the liposomescan be made to bind to specific sub-populations of cells.

In still another embodiment, the therapeutic E2 protein can be deliveredby way of an artificial viral envelope (AVE). Briefly, the art asdescribed a number of viral envelopes which exploit molecularrecognition of cell surface receptors by viral surface proteins as ameans for selective intracellular delivery of macromolecules, includingproteins. According to the method of Schreier, et. al., U.S. Pat. No.5,252,348, a virtually unlimited number of artificial viral envelopescan be prepared and applied using recombinant or isolated surfacedeterminants. For example, the AVEs be generated as viral mimetics of anumber of human viruses including arboviruses; flaviviridae;bunyaviridae; hepatitis viruses; Epstein-Barr viruses; herpes viruses;paramyxoviruses; respiratory syncytial virus; retroviruses includinghuman T-lymphotrophic virus type I and II (HTLV-I/II) and humanimmunodeficiency virus type 1 and 2 (HIV-1/2); rhinoviruses;orthopoxviruses; and human papilloma viruses.

In another embodiment, direct delivery of a therapeutic E2 protein maybe facilitated by chemical modification of the polypeptide itself. Onesuch modification involves increasing the lipophilicity of the E2protein in order to increase binding to the cell surface, in turn,stimulating non-specific endocytosis of the protein. Lipophilicity maybe increased by adding a lipophilic moiety (e.g., one or more fatty acidmolecules) to the E2 protein. A wide variety of fatty acids may beemployed. For example, the protein may be palmitoylated. Alternatively,a lipopeptide may be produced by fusion or cross-linking, to permit theE2 protein to resemble the natural lipopeptide from E.coli,tripalmitoyl-S-glycerylcysteil-seryl-serine, at its amino terminus. Thislipopeptide has been shown to increase the uptake of fused peptides (P.Hoffmann et al., (1988) Immunobiol. 177:158-70). Lipophilicity may alsobe increased by esterification of the protein at tyrosine residues orother amino acid residues. And uptake of the E2 protein may be increasedby addition of a basic polymer such as polyarginine or polylysine (Shenet al. (1978) PNAS 75:1872-76).

Direct delivery of E2 proteins according to this invention may also beeffected by the use of transport moieties, such as protein carriersknown to cross cell membranes. For example, an E2 protein may be fusedto a carrier protein, preferably by a genetic fusion which may beexpressed in a system such as E.coli or yeast. According to oneembodiment of this invention, the amino terminus of the E2 protein maybe fused to the carboxy terminus of a transport moiety using standardtechniques.

Nucleotide sequences encoding such carrier-E2 protein fusion proteins,operatively linked to regulatory sequences, may be constructed andintroduced into appropriate expression systems using conventionalrecombinant DNA procedures. The resulting fusion protein may then bepurified and tested for its capacity to (1) enter intact eukaryoticcells and (2) inhibit E2-dependent gene expression and viral DNAreplication once inside the intact eukaryotic cells.

In choosing a useful carrier protein, those of skill in the art willrecognize the desirability of appropriate control experiments designed,inter alia, to test the possibility that the carrier portion of thefusion protein itself interacts with elements of the E2 transcriptionalregulation system. If the carrier portion of the fusion protein is foundto have undesirable interactions, such as activation of E2-dependentenhancer elements, the portions of the carrier sequence responsible forthese interactions should be identified and deleted in a way whichpermits the sequence to retain its carrier capacity. Alternately, one ofseveral conventional carrier sequences which do not interact withelements of the E2 transcriptional regulation system can be substituted.

Useful carrier proteins include, for example, bacterial hemolysins or“blending agents”, such as alamethicin or sulfhydryl activated lysins.Other carrier moieties which may be used include cell entry componentsof bacterial toxins, such as Pseudomonas exotoxin, tetanus toxin, ricintoxin, and diphtheria toxin. Also useful is melittin, from bee venom.Other useful carrier proteins include proteins which are viralreceptors, cell receptors or cell ligands for specific receptors thatare internalized, i.e., those which cross mammalian cell membranes viaspecific interaction with cell surface receptors, recognized and takeninto the cell by cell surface receptors. Such cell ligands include, forexample, epidermal growth factor, fibroblast growth factor, transferrinand platelet-derived growth factor. Alternatively, the ligand may be anon-peptide, such as mannose-6-phosphate, which permits internalizationby the mannose-6-phosphate receptor. The transport moiety may also beselected from bacterial immunogens, parasitic immunogens, viralimmunogens, immunoglobulins or fragments thereof that bind to targetmolecules, cytokines, growth factors, colony stimulating factors andhormones. A transport moiety may also be derived from the tat protein ofHIV-1.

As an alternative or addition to the above-described chemicalmodifications and protein carriers, which may be employed alone or incombination, other agents which allow penetration of the keratinizedcell layer may be employed to facilitate delivery of the E2 proteins ofthis invention to papillomavirus-infected cells. In topicalapplications, for example, the E2 protein may be administered incombination with dimethylsulfoxide, an agent which promotes penetrationof cell membranes by substances mixed with it. Useful keratinolyticagents include, for example, salicylic acid, urea, and alpha-hydroxyacids. For such applications, the E2 protein and any other agentmay be administered topically, in cream or gel form.

According to an alternate embodiment of this invention, the therapeuticE2 protein may be administered serially or in combination with othertherapeutics used in the treatment of papillomavirus infections ordiseases caused by them. Such therapeutics include interferons, such asIFN-γ, IFN-α and IFN-β derived from natural sources or produced byrecombinant techniques, other cell mediators formed by leukocytes orproduced by recombinant techniques such as for example, interleukin-1,interleukin-2, tumor necrosis factor, macrophage colony stimulatingfactor, macrophage migration inhibitory factor, macrophage activationfactor, lymphotoxin and fibroblast growth factor. Alternatively, the E2protein may be administered serially or in combination with conventionaltherapeutic agents or regimens such as, for example, salicylic acid,podophyllotoxin, retinoic acid, surgery, laser therapy and cryotherapy.Such combination therapies may advantageously utilize less thanconventional dosages of those agents, or involve less radical regimens,thus avoiding any potential toxicity or risks associated with thosetherapies.

It will also be understood by those skilled in the art that any of theabove enumerated delivery methods may be augmented, where topicalapplication is being carried out, by the use of ultrasound oriontophoretic delivery devises which facilitate transdermal delivery ofproteins. See, for example, Banga et al. (1993) Pharm Res 10:697-702;and Mitragotri et al. (1995) Science 269:850-853.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Carcinogenic progression of a human papillomavirus (HPV)-infected cellis often associated with integration of the viral genome in a mannerwhich results in the loss of expression of the viral regulatory proteinE2. One function of E2 is the regulation of expression of the viraloncogenes, E6 and E7. Introduction of the bovine papillomavirus type 1(BPV-1) E2 transactivator (E2-TA) in HeLa cells, an HPV type 18(HPV-18)-positive cervical carcinoma cell line results in growth arrest.In this study, we have found that the HPV-16 and HPV-18 E2 proteinsshare with BPV-1 E2-TA the ability to suppress HeLa cell growth. Thisproperty was not observed for the BPV-1 E2 transcriptional repressor(E2-TR). Analysis of various mutant E2 proteins for growth suppressionrevealed a requirement for the intact transactivation and DNA bindingdomains. A HeLa cell line (HeLa-tsE2) which expressed a conditionalmutant E2 protein that was functional only at the permissive temperature(32° C.) was established, permitting an analysis of the molecular andcellular consequences of E2 expression. Our data indicates that onemechanism by which E2 suppresses cellular growth is through repressionof E6 and E7 expression, thereby enabling the cellular targets of E6 andE7 to resume regulation of the cell cycle.

The functions of the E2 proteins have been extensively characterized forBPV-1. The BPV-1 E2 ORF encodes three different proteins: an internallyinitiated E2 transcriptional repressor (E2-TR), a spliced productbetween E8 and E2 (E8-E2), and the E2 transactivator (E2-TA) (Hubbert etal. (1988) Proc. Natl. Acad. Sci. USA, 85:5864-5868; Lambert et al.(1989) J. Virol., 63:3151-3154). The full-length E2 protein (E2-TA) canfunction as a transactivator or a repressor, depending on the locationof E2 binding sites in a responsive promoter. It has two functionaldomains which are well conserved among the E2 proteins of differentpapillomaviruses (Giri and Yaniv (1988) EMBO J., 7:2823-2829; McBride etal. (1991) J. Biol. chem., 266:18411-18414), a 200-amino-acidtransactivation domain at the N terminus and a 90- to 100-amino-acidC-terminal domain that is essential for dimerization and DNA binding (Liet al. (1989) Genes Dev., 3:510-526; McBride et al. (1989) Proc. Natl.Acad. Sci. USA, 86:510-514, McBride et al. (1988) EMBO J., 7:533;Moskaluk and Bastia (1988) J. Virol., 62:1925-1931). A flexible spacerregion called “the hinge” separates the transactivation and DNA bindingdomains. Both E2-TR and E8-E2 can inhibit the transactivation functionof the full-length protein, through mechanisms that may involvecompetition with E2-TA for DNA binding or by subunit mixing andheterodimer formation with the full-length protein. E2-TA can directlyrepress transcription of promoters in which the E2 binding sites arelocated near essential promoter elements such as the TATA box or SP1binding sites (Dostatni et al. (1991) Genes Dev., 5:1657-1671; Gloss andBernard (1990) J. Virol., 64:5577-5584; Matsushime et al. (1994) Mol.Cell Biol., 14:2066-2076; Li et al. (1991) Cell 65:493-505, Romanczuk etal. (1990) J. Virol., 64:2849-2859; Spalholz et al. (1991) J. Virol.,65:743-753; Thierry and Howley (1991) New Biol., 3:90-100; Vande Pol andHowley (1990) J. Virol., 64:5420-5429). In these cases, E2-TA isbelieved to interfere with the assembly of the transcriptionalinitiation complex.

The studies presented here were designed to examine the effect of E2expression in cervical carcinoma cell lines. We were able to confirmearlier studies showing that expression of BPV-1 E2-TA results in growthsuppression of HeLa cells. However, we found that (i) the expression ofHPV E2 proteins specifically inhibited PV-infected and PV-transformedcells, as opposed to the non-specific inhibition by BPV E2 proteinsshown in the art, and (ii) the E2-TR protein was unable to suppresscellular growth. Using a series of mutated E2 proteins, were able todetermine which domains of the E2 protein were important for thisactivity. We were also able to demonstrate that HPV-16 and HPV-18 E2proteins had similar growth suppression functions and these proteinsmust maintain transcriptional activation function in order to retainthis growth suppression function although transcriptional activationfunction alone is not sufficient (e.g, VP16 E2). Establishment of a HeLacell line allowed further characterization of E2-mediated growthsuppression. These studies indicate that at least one consequence of E2expression in HPV-positive cell lines is decreased expression of theviral oncoproteins, resulting in cell cycle arrest.

Finally, these studies also exemplify the preparation and isolation oftwo different altered E2 polypeptides from two different strains ofpapillomavirus, i.e., bovine and human, that have the advantage of beingable to suppress cell growth without triggering undesired papillomavirusreplication.

Materials and Methods

Recombinant plasmids: The plasmids used for expression of the variousBPV-1 E2 proteins were based on C59, which utilizes the SV40 earlypromoter to express the full-length E2 protein (Spalholz et al. (1991)J. Virol., 65:743-753; Yang et al. (1985) Nature (London) 318:575-577).The C59-derived E2-TA plasmid used in this study has been modified tocontain a Kozak initiation consensus sequence (CCACCATG [Kozak, M.(1991) J. Biol. Chem., 266:19867-19870]) as previously described(Winokur and McBride (1992) EMBO J., 11:4111-4118). The plasmids usedfor expression of E2-TR and the E2-TR with the translation terminationlinker in E5 (p1175) were previously described (Lambert et al. (1987)Cell 50:68-78). Plasmids which express the E2 mutant proteinsE2_(>1-15), E2_(>157-282), E2_(>220-309) (McBride et al. (1989) Proc.Natl. Acad. Sci. USA, 86:510-514; Winokur and McBride (1992) EMBO J.,11:4111-4118), and E2₁₋₂₁₈ have been previously described (Yang et al.(1985) Nature (London) 318:575-577). Plasmids that express E2 mutantproteins E2_((E39A)) and E2_((173A)) were constructed as described inSakai et al. (1996) J. Virology 70:1602-1611.

ts E2 was subcloned from plasmid pE2ts-1 (DeMaio and Settleman (1988)EMBO J., 7:1197-1204) by PCR using oligonucleotides containing a HindIIIsite at the 5′ end and an XbaI site at the 3′ end of the E2 gene. Pfupolymerase (from Stratagene) was used to ensure high fidelity. The DNAfragment containing temperature-sensitive E2 (tsE2) was cloned into thevector pRC-CMV (Invitrogen) at the HindIII-XbaI sites of the polylinker.This vector also contains the gene for neomycin resistance. The sequenceof this clone was verified by automated DNA sequence analysis (ABI model373A sequencer). The mutant E2 gene contains an insertion of thefour-amino-acid sequence Pro-Arg-Ser-Arg between amino acids 181 and 182(DeMaio and Settleman (1988) EMBO J., 7:1197-1204).

Cell culture: HeLa, HT-3, SiHa, Caski, and C33A are human cervicalcancer cell lines obtained from the American Type Culture Collectionthat have been previously analyzed for the presence of HPV DNA and HPVRNA (Yee et al. (1985) Am. J. Pathol., 119:361-366). Saos-2, a humanosteosarcoma cell line, was obtained from Phil Hinds (Harvard MedicalSchool). An immortalized human foreskin keratinocyte cell line (W16) wasimmortalized by using the full-length genome of HPV-16 linearized withBamHI, as previously described (Romanczuk and Howley (1992) Proc. Natl.Acad. Sci. USA, 89:3159-3163).

HeLa, SiHa, Caski, C33A, and Saos-2 cells were maintained in Dulbeccomodified Eagle medium with 10% fetal bovine serum. HT-3 cells weremaintained in McCoy's 5A medium (Gibco/BRL) with 10% fetal bovine serum.The HPV-16-immortalized Keratinocyte line (W16) was maintained in 3+1medium (3 parts KGM plus 1 part Dulbecco modified Eagle medium).

Growth suppression assay. Cells were seeded at 1×10⁶ to 2×10⁶ cells per10-cm-diameter dish the day before transfection. Cells were transfectedby lipofection (Lipofectin; GIBCO BRL), using 8 to 10 μg of plasmid DNApurified through two CsCl gradient centrifugations per 10-cm-diameterplate. Sixteen hours after transfection, cells were referred; at 24 hposttransfection, the cells were split and placed under selection inmedium containing 10% fetal bovine serum and G418 (concentrationdependent on the cell line). Cells were maintained under selection for 2to 3 weeks until the number of drug-resistant colonies could bedetermined. Cells were fixed in 10% formaldehyde for 15 min. washed, andstained with methylene blue for 15 min. Plates were washed, andG418-resistant colonies were counted.

Chloramphenicol acetyltransferase assays: Reporter plasmid wascotransfected into HeLa cells (Chen and Okayama (1987) Mol. Cell Biol.,7:2745-2752) with the indicated expression plasmids (empty vector wasused to normalize the amount of total DNA transfected) by the calciumphosphate method. At 48 h after transfection, cells were harvested andlysed in 250 mM Tris (pH 8.0), and chloramphenicol acetyltransferaseactivity was determined (Gorman et al. (1982) Mol. Cell Biol.,2:1044-1051).

Replication Assay: Transient replications of HPV16 origin-containingplasmid p16Ori were analyzed essentially by the technique previouslydescribed by Del Vecchio et al. (Del Vecchio et al. (1992) J. Virol.66:5949-5958). At 70 h after transfection, low-molecular weight DNA wasextracted by the method of Hirt (Hirt, (1967) J. Mol. Biol. 26:365-369)modified as described below. Plates were washed two times withphosphate-buffered saline without magnesium or calcium. The cells werethen scraped into a 1.5-ml tube, pelleted, resuspended in 200 μl ofbuffer 1 (50 mM glucose, 25 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mg ofRNase A per ml), and lysed by the addition of 200 μl of buffer II (1%SDS, 0.2 N NaOH). After 5 min, 200 μl of ice-cold buffer III (3 M to 5 Mpotassium acetate) was added, and the samples were placed at 4° C. forat least 1 h. After centrifugation for 10 min at 4° C., they wereextracted once with buffer-saturated phenol and once withphenol-chloroform-isoamyl alcohol and precipitated with ethanol. Todistinguish replicated DNA from input DNA, the extracted DNA sampleswere digested with DpnI. (Plasmid DNA was prepared from a Dammethylase-positive bacterial strain, rendering it sensitive to DpnIdigestion.) To linearize p160ri, the sample DNAs were also digested withXmnI. The digested samples were separated by 1.0% agarose gelelectrophoresis and then analyzed by Southern blotting. The alkalinetransfer to Hybond−N+ membranes (Amersham) was performed by themanufacturer's method. A DNA fragment encompassing the origin sequenceand lacZ coding sequence was generated for use as a probe by PCR withthe p160ri template. This fragment was labeled by using a random-primedlabeling kit (Stratagene). Hybridization in 50% formamide-containingbuffer and subsequent washes were done by standard methods (Sandler, etal. (1993) J. Virol. 67:5079-5087). The sample DNA digested with DpnIand XmnI was also subjected to PCR-southern blot analysis. To amplifyDpnI digestion-resistant (replicated) DNA, primers (nt 1814 to 1843 and2628 to 2657 of pKS(-) BluescriptII; Strategene; GenBank accessionnumber X52329) were used with 10 cycles of PCR amplification. There arenine DpnI sites within the sequence amplified with these primers. Theamplified DNA was analyzed by Southern blotting. A Phosphor-Imager(Bio-Rad Laboratories) was used for quantitation of the hybridizedsignals. The amount of the template DNA for amplification was confirmedto be within the quantitative range (input DNA=0.1 to 50 pg).

Generation of HeLa-ts E2 cell line: ts E2 was linearized with BglII andtransfected into HeLa cells by lipofection. Transfected cells containingts E2 were selected for in 450 μg of G418 per ml. and individualcolonies were analyzed.

RNA analysis: The HeLa-tsE2 cell line and the control cell linecontaining the vector alone (HeLa-V) were split, and 10⁶ cells wereplated. Cultures were at maintained at 32 or 38° C. for 5 days. TotalRNA was harvested by using a Biotex RNA isolation kit. Total RNA (14 μg)was separated by 1.2% agarose-formal-dehyde gel electrophoresis forNorthern (RNA) blot analysis. The RNA was transferred to a GeneScreenPlus filter and hybridized with a probe for HPV-18 E6/E7 mRNA levels andwith the cyclophilin probe (Danielson et al. (1988) DNA, 7:261-267).

Immunological procedures: For immunoblotting, cell lysates were preparedin lysis buffer (0.1 M NaCl, 2mM EDTA, 20 mM Tris [pH 8.0], 1% NonidetP-40, per ml, 5 mM sodium fluoride, 1 mM sodium orthovanadate) on icefor on ice for 30 min. Lysates were cleared by centrifugation at15,000×g at 4° C. for 5 min. A 100 μg aliquot of protein from the celllysate of each sample was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed byimmunoblotting (Towbin et al. (1979) Proc. Natl. Acad. Sci. USA,86:4350-4354). The protein concentrations were determined by the Bio-Radprotein assay. Monoclonal antibody 1801 (Ab-2 from Oncogene Science) wasused for p53 detection. Monoclonal antibody 245 (PharMingen) was usedfor pRb detection. Mouse monoclonal antibody sdi-1p21 (1509-A;PharMingen) was used for p21/WAF1/C1p1 detection. Horseradishperoxidase-conjugated rabbit anti-mouse antibody (NA 931; Amersham) wasused, followed by enhanced chemiluminescence detection (Renaissancesystem; NEN).

Immunocomplex kinase assays: Immunoprecipitations were carried outessentially as described previously (Matsushime et al. (1994) Mol. CellBiol., 14:2066-2076), using 100 μg of cellular extract. Rabbitpolyclonal antibody C-8 (Santa Cruz) was used to precipitate cyclin A.Mouse monoclonal antibody HE-111 (Santa Cruz) was used to precipitatecyclin E complexes.

Kinase assays were carried out as described previously (Matsushime etal. (1994) Mol. Cell Biol., 14:2066-2076), using histone H1 as asubstrate. Proteins were separated by SDS-PAGE (11% polyacrylamide gel).Coomassie blue stained, and dried, and phosphorylated proteins werevisualized by autoradiography.

Results

(i) Suppression of HeLa Cell Growth by BPV-1 E2-TA but Not E2-TR.

Expression of the full-length BPV-1 E2-TA protein in HeLa cells resultsin suppression of cell growth as assayed by a reduction in colonyformation (Thierry and Yaniv (1987) EMBO J., 6:3391-3397). We believethat the decreased expression of the viral oncoproteins may contributeto this growth suppression, since expression of the full-length E2proteins of BPV-1, HPV-16, and HPV-18 as well as HPV-18C (anartificially truncated HPV-18 E2 repressor construct, analogous to BPV-1E2-TR) is capable of down regulating transcription from the HPV-18 P₁₀₅promoter (Thierry and Howley (1991) New Biol., 3:90-100). Furthermore,growth suppression of HeLa cells by BPV-1 E2-TA expressed from anSV40-based vector results in the down regulation of E6 and E7 expressionfrom the viral P₁₀₅ promoter (Hwang et al. (1993) J. Virol.,67:3720-3729).

To further investigate the mechanisms involved in the E2 regulation ofcellular proliferation, we examined whether BPV-1 E2-TR could suppressHeLa cell growth like the full-length BPV-1 E2-TA. HeLa cells werecotransfected with a plasmid conferring neomycin resistance (pSV2neo)and a plasmid encoding either BPV-1 E2-TA or BPV-1 E2-TR. Cells weresplit after transfection and maintained in G418-containing medium for 14to 21 days, at which time the number of G418-resistant colonies wasdetermined. No growth suppression was observed with BPV-1 E2-TR (FIG.1). Since the E2-TR plasmid used in this experiment contains an intactE5 gene which might mask or otherwise affect a growth-suppressive effectof E2-TR, an E2-TR construct (p1175) with a translation terminationlinker in E5 was tested and also found to be defective for HeLa cellgrowth suppression. Similar results were obtained when puromycinselection rather G418 selection was used, indicating that these resultswere not specific for neomycin resistance selection.

The lack of growth suppression by E2-TR was unexpected, since E2-TR hasbeen shown to specifically repress E2-responsive promoters. To confirmthat E2-TR was indeed expressed in HeLa cells, the E2-TR plasmid wastested for its ability to repress E2-TA transactivation in HeLa cells.HeLa cells were transfected with the E2-TA plasmid, the E2-responsiveplasmid 6XE2TKCAT (Thierry et al. (1990) Mol. Cell Biol., 10:4431-4437),and increasing amounts of the E2-TR plasmid. We observed that E2-TR wasable to specifically repress E2-TA transactivation, indicatingexpression of functional E2-TR in HeLa cells.

(ii) HPV-16 and HPV-18 E2 Proteins Suppress Cell Growth in HeLa CervicalCarcinoma Cells.

The abilities of HPV-16 and HPV-18 E2 proteins to suppress HeLa cellgrowth were next examined. In this series of experiments, we testedwhether the E2 proteins of the high-risk HPVs shared thegrowth-suppressive function shown for the BPV-1 E2-TA protein. Theexperiment was performed with HPV-16 E2 and HPV-18 E2 expressionplasmids that had been previously shown to express functional E2 proteincapable of supporting origin-dependent DNA replication and of activatingE2-responsive promoters (Del Vecchio et al. (1992) J. Virol.,66:5949-5958). Both HPV-16 E2 and HPV-18 E2 proteins suppressed HeLacell growth in the colony formation assay (Table 1). This result differsfrom a previous report that expression of HPV-18 E2 was not able tosuppress the growth of HeLa cells (Thierry and Yaniv (1987) EMBO J.,6:3391-3397).

TABLE 1 HeLa cell growth suppression by HPV-16 and HPV-18 E2^(a) No. ofG418-resistant colonies^(b) Transfection Expt. 1 Expt. 2 HPV-16 E2  0  0HPV-18 E2  0  0 Vector alone 34 41 ^(a)HeLa cells were transfected with1 μg of pSV2neo and 10 μg of either HPV-16 E2- or HPV-18 E2-expressingplasmid or vector alone. ^(b)Transfected cells were maintained underselection in medium containing 500 μg of G418 per ml for 2 to 3 weeks,and the number of G418-resistant colonies from each transfection wasdetermined.(iii) Growth Suppression Mediated by E2 is Observed in OtherHPV-Positive Cell Lines.

To examine the specificity of E2 growth suppression, additional celllines were assayed for their responses to E2 expression using the colonyreduction assay. E2 growth suppression was observed in each of the threeHPV-positive cell lines tested in which the viral genes are expressedfrom the viral long control region. These include two HPV-16-positivecervical carcinoma cell lines. SiHa and Caski, and W16, which is a humanforeskin keratinocyte cell line immortalized by HPV-16 (Table 2). Growthsuppression by BPV-1 E2-TA was observed in each of these cell lines, andno growth suppressive effect was observed with the BPV-1 E2-TR protein.These data are in agreement with the results of Hwang et al. (Hwang etal. (1993) J. Virol., 67:3720-3729), who also reported growthsuppression of other HPV-positive cervical cancer cell lines by BPV-1E2-TA.

TABLE 2 BPV-1 E2 growth suppression of HPV-16-expressing human celllines^(a) No. of G418-resistant colonies SiHa Caski W16 TransfectionExpt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 E2-TA 6 4 2 0 4 8 E2-TR52 42 59 67 18 19 Vector alone 46 57 51 55 24 23 ^(a)Indicated celllines were transfected with 1 μg of pSV2neo and 10 μg of either BPV-1E2-TA- or BPV E2-TR-expressing plasmid or vector alone.

To determine whether E2 can act as a general growth suppressor, twocervical carcinoma cell lines which are HPV negative (C33A and HT-3) anda human osteosarcoma cell line (Saos-2) were tested for growthsuppression by E2-TA. It was previously reported that E2-TA couldsuppress the growth of HT-3 cells when introduced by infection with anE2-expressing recombinant SV40 (Hwang et al. (1993) J. Virol.,67:3720-3729). In our assay, however, the growth of each of theseHPV-negative cell lines, including HT-3, was unaffected by expression ofthe E2-TA protein (Table 3). Thus, the E2 growth suppression in thecolony reduction assay is specific for HPV-positive cells.

TABLE 3 Lack of BPV-1 E2 growth suppression of HPV-negative human celllines^(a) No. of G418-resistant colonies Saos-2 Transfection Expt. 1Expt. 2 HT-3 E2-TA 67  80 24 E2-TR 81 102 28 Vector alone 62  99 19^(a)See the footnote to Table 2. For C33A cells, values were >500 in allassays in two experiments.(iv) Intact DNA Binding and Transactivation Functions of the E2 Proteinare Required for Inhibition of HeLa Cell Growth.

The domains of BPV-1 E2 necessary for growth suppression were mapped byusing a series of truncation and deletion mutants of E2, which had beenpreviously characterized for their transactivation and replicationfunctions (Winokur and McBride (1992) EMBO J., 11:4111-4118). Thegrowth-suppressive phenotype conferred by the E2 deletion mutants wasassayed in HeLa cells by using the colony reduction assay (FIG. 3A).Growth suppression was observed with E2-TA and with E2_(>220-309), whichis a mutant deleted of a large segment of the hinge region butcontaining an intact transactivation domain and an intact DNAbinding/dimerization domain. No growth suppression was observed foreither E2_(>1-15) or E2_(>157-220), both of which are deficient for thetranscriptional transactivation and DNA replication functions inmammalian cells. Each of these E2 mutants can inhibit thetransactivation properties of E2-TA in trans. An E2 mutant protein(E2₁₋₂₁₈) consisting of only the transactivation domain similarly had nonegative effect on cell growth. The E2_(>220-309) mutant, which hasgrowth-suppressive activity, is able to transactivate an E2-responsiveplasmid but is defective in origin-dependent DNA replication (Winokurand McBride (1992) EMBO J., 11:4111-4118). We confirmed thetransactivation or transrepression properties of each of these mutant E2proteins in HeLa cells. The expression of the E2 proteins wasdemonstrated in COS cells by immunoblotting, using expression fromrecombinant PAVA viruses. In these studies, it was noted that theE2₁₋₂₁₈ mutant was expressed at a somewhat lower level than the other E2proteins. These results indicate that the BPV-1 E2 growth-suppressiveeffect in HeLa cells requires a functional transactivation domain aswell as an intact DNA binding domain but that an intact DNA replicationfunction is apparently not necessary.

The genetic organization of BPV-1 is complex, with overlapping ORFs atthe 3′ end of the early region, raising the possibility that another ORFcontributes to the growth suppression phenotype. This, however, is quiteunlikely. The intact E2-TA expression vector that was used containsadditional ORFs, including E3 (3267 to 3551), E4 (3173 to 3526), and E5(3714 to 4010). The E2_(>220-309) deletion mutant plasmid, whichexpresses an E2 capable of growth suppression, is deleted of most of thecoding regions of E3 and E4 (from 3265 to 3532 of the BPV-1 genome).Furthermore, E5 has been previously shown not to be associated with thegrowth-suppressive effect observed with the BPV-1 E2 expression vector(Thierry and Yaniv (1987) EMBO J., 6:3391-3397). Thus, we conclude thatthe growth-suppressive phenotype can be attributed to E2.

We next examined the specificity of the E2 transactivation domain insuppressing HeLa cell proliferation by testing whether this functioncould be provided by other acidic activation domains. Chimericconstructs containing the E2 DNA binding/dimerization domain fused toeither the acidic transactivation domain of the herpesvirus VP16transcriptional activator or the spi oncogene (Gauthier et al. (1993)EMBO J., 12:5089-5096) were tested in the colony reduction assay (FIG.3B). Although both chimeric proteins were able to efficientlytransactivate E2-responsive reporter constructs in HeLa cells, neitherhad the ability to suppress HeLa cell proliferation. This findingindicates that the ability of the E2 transactivation domain to suppresscellular proliferation is specific and not due to its generaltransactivation property.

The dimerization and DNA binding properties of BPV-1 E2 have beenseparated by point mutations in the DNA binding domain (Barsoum et al.(1992) J. Virol., 66:3941-3945; McBride et al. (1989) J. Virol.,63:5076-5085). E2 mutants that can dimerize but not bind DNA and thatcan no longer dimerize or bind DNA have been described. Two of these E2mutants were tested for the ability to suppress HeLa cell growth.Neither the E2_((I331R)) mutant, which is defective for bothdimerization and DNA binding, nor the E2_((R344K)) mutant, which candimerize but not bind DNA, was capable of suppressing HeLa cellproliferation (FIG. 3B). These results suggest that both the E2transactivation and DNA binding functions are necessary for the E2growth suppression phenotype.

(v) Molecular Consequences of E2 Expression.

To study the downstream effects of E2 expression in HPV-positive cells,we used a conditional BPV-1 E2 mutant that has normal replication andtransactivation functions at 32° C. which are inactivated at either 37or 39° C. (DiMaio and Settleman (1988) EMBO J., 7:1197-1204). Thismutant E2 gene was subcloned behind the cytomegalovirus promoter into anexpression vector containing the neomycin resistance gene to generateplasmid pRC-tsE2. This plasmid was introduced into HeLa, SiHa, and C33Acells, and the number of G418-resistant colonies was determined after 21days at 32 and 38° C. (Table 4). Following transfection at thenonpermissive temperature (38° C.), the cells were split and incubatedat either 32 or 38° C. under G418 selection. At the permissivetemperature (32° C.), tsE2 inhibited colony formation, similar to theeffect of the wild-type E2-TA, in both the HeLa and SiHa cell lines.However, at the nonpermissive temperature (38° C.), there was no growthinhibition in any of the cell lines tested. In agreement with theresults obtained with wild-type E2, no growth suppression was observedwith tsE2 in C33A cells at the permissive temperature. Controltransfections performed with the pRc-neo vector alone confirmed that thegrowth suppression observed in HeLa and SiHa cells was not due totemperature, since G418-resistant colonies appeared for both cell linesat 32°C.

TABLE 4 Temperature-sensitive growth phenotype of cells containing theE2 mutant^(a) No. of G418-resistant colonies pRC-tsE2 pRC-neo Cell line32° C. 38° C. 32° C. 38° C. HeLa 0 73 41 61 SiHa 0 86 37 77C33A >500 >500 >500 >500 ^(a)Indicated cell lines were transfected with1 μg of pSV2neo and 10 μg of either BPV-1 tsE2 or vector alone.

pRC-tsE2 was used to generate a G418-resistant HeLa cell line at 38° C.which expressed the conditional E2 mutant protein (HeLa-tsE2). HeLa-tsE2clonal lines were characterized for the ability to transactivate anE2-responsive plasmid and for their growth properties at 32 and 38° C.As a control, a G418-resistant HeLa cell line (HeLa-V) was alsoestablished by using the vector without insert. The HeLa-tsE2 cells weregrowth arrested at 32° C., whereas the HeLa-V cells were not.Fluorescence-activated cell sorting analysis indicated that theHeLa-tsE2 cells were arrested in G₁ when grown at 32° C., consistentwith previously reported data indicating that BPV-1 E2 expression HeLacells from a recombinant SV40 leads to a G₁ cell cycle arrest (Hwang etal. (1993) J. Virol., 67:3720-3729). The HeLa-tsE2 cell line wascharacterized for HPV-18 RNA levels. At the permissive temperature, thelevel of E6/E7 message was at least three- to fourfold lower than inHeLa-tsE2 cells at 38° C. There was a temperature-dependent decrease of1.5-fold in the HeLa-V cell line at 32° C. compared with HeLa-V cellsgrown at 38° C.

(vi) Effects of E2 Expression on Cellular Proteins in HeLa Cells.

The E2-dependent loss of E6/E7 message and growth arrest may be mediatedthrough cellular targets of the E6 and E7 proteins. Since HPV-18 E6promotes the degradation of p53, the decrease in expression of theendogenous viral mRNA would be predicted to result in lower levels of E6and increased levels of p53. The levels of p53 were examined in theHeLa-tsE2 cell line following a shift to the permissive temperature.Immunoblot analysis of total cell extracts demonstrated a 20-foldincrease in the level of p53 in HeLa-tsE2 cells shifted to 32° Comparedwith normal HeLa cells or HeLa-tsE2 cells grown at 38° C. A decrease inp53 levels was noted in HeLa-V cells at 32° C. The reason for thislowered level of p53 is not known, but this finding suggests that thedestabilizing effect of E6 might be even greater than the 20-fold changein the HeLa-tsE2 cell line.

Induction of the cyclin-dependent kinase inhibitor p21/WAF1/Cip1/Sdi1occurs in response to increased levels of p53 (Eldiery et al. (1993)Cell, 75:817-825). An increased level of p21 protein was observed in theHeLa-tsE2 cells after a shift to the permissive temperature (32° C.).Notably, in the HeLa-V cell line at 32° C., which had very low levels ofp53, no p21 was detected.

To examine whether the increased levels of p21 in the HeLa-tsE2 cellline at the permissive temperature resulted in an inhibition of thecyclin-dependent kinases, cell lysates were prepared andimmunoprecipitated with cyclin-specific antibodies, and kinase assayswere performed with histone H1 as a substrate (Matsushime et al. (1994)Mol. Cell Biol., 14:2066-2076). Cyclin E is associated with Cdk2 and isactive in late G₁ (Dulic et al. (1993) Proc. Natl. Acad. Sci. USA,90:11034-11038; Koff et al. 1992) Science, 257:1689-1694). Inhibition ofthe cyclin E-associated kinase activity was observed in the HeLa-tsE2cells at the permissive temperature. The cyclin E-associated kinase wasactive in the HeLa-tsE2 cells at 38° C. and in the HeLa-V cells at both32 and 38° C. Cyclin A-associated kinase activity was also inhibited inthe HeLa-tsE2 cells at the permissive temperature but not at thenonpermissive temperature or in the HeLa-V cell line.

An immunoblot analysis of pRB was performed on extracts from theHeLa-tsE2 and HeLa-V cell lines grown at 32 or 38° C. Both thehyperphosphorylated and hypophosphorylated forms of pRB were detected ineach case; however, the pRB forms in the HeLa-tsE2 cell line shifted to32° C. were predominantly hypophosphorylated. There was littledifference in the phosphorylation state of pRB in the HeLa-tsE2 cellsgrown at 38° C. and pRB in the HeLa-V control cell line at eithertemperature.

(vii) Effects of E2 Expression (HPV16) on Growth Suppression,Transcription, and Virus Replication

The above studies were extended with an E2 polypeptide from a high riskhuman strain of papillomavirus (HPV16). In this study, astructure-function analysis of the N-terminal domain of HPV 16 E2protein was performed. Three different biological activities wereexamined for the HPV 16 E2 protein: transcriptional transactivation(which indicates the ability of the E2 polypeptide to repress viraltranscription, see, e.g., Goodwin et al. (1998) J. Virology72:3925-3934), enhancement of origin-dependent DNA replication, andrepression of cell growth. By targeting highly conserved amino acids foralanine substitution mutagenesis, mutations were identified in theN-terminal domain that can disrupt either the transactivation or DNAreplication functions of HPV16 E2 (see, e.g., E2 (E39A) and E2 (173A);Table 5 and FIG. 4). These observations indicate that these functionsare distinct and separable. Furthermore, the binding capacity of E2 forE1 was observed to be critical for its function in origin-dependent DNAreplication. Importantly, these studies allowed for the identificationof an E2 mutant that can repress cell growth while avoiding thetriggering of undesired viral replication (e.g., HPV E2 (E39A)).

TABLE 5 HPV E2 (E39A) represses cell growth but does not promote viralreplication DNA Transcriptional replication activation Number ofColonies E2 Construct function function Expt. 1 Expt. 2 Expt. 3 Vector −− 720 524 532 BPV E2 + + 19 40 5 BPV E2TR − − 632 336 642 HPV 16E2 + +92 96 29 HPV 16E2 − + 86 11 25 E39A HPV 16E2 + − 170 404 119 173A

In conclusion, this analysis of the HPV 16 E2 protein by alaninesubstitution mutagenesis of conserved amino acid residues in the Nterminus has provided evidence that E2 is a multifunctional proteinwhose different activities can be separated. The strong transcriptionalactivation function of E2 can be dissociated from its ability to enhanceE1-mediated, origin-specific DNA replication. The DNA replicationfunction of E2 appears to depend on its ability to bind E1. Importantly,the above studies demonstrate that it is feasible to engineer E2polypeptides defective for promoting viral replication and yet able tosuppress cell growth and this is clearly exemplified by, e,g., thismutant, as well as the BPV mutant E2_(Δ220-309) (see FIG. 4). Theisolation of an E2 polypeptide defective for viral replication but ableto suppress cell growth, e.g., in papillomavirus-infected cells hasimportant therapeutic advantages. For example, such a polypeptide may beused to suppress growth in papillomavirus-infected cells without thedanger of triggering virus replication in the cell.

Discussion

The E2 transactivation and DNA binding/dimerization domains were eachnecessary for E2-mediated growth suppression of HeLa cells.E2_(>220-309), which is defective in the replication function butretains the ability to transactivate an E2-responsive plasmid (Winokurand McBride (1992) EMBO J., 11:4111-4118), was able to suppress growthof HeLa cells, indicating that the growth-suppressive properties of E2can be unlinked from its DNA replication properties. These sameactivities where also achieved using a altered E2 polypeptide from HPV16by introducing a single amino acid substitution into the transactivationdomain (i.e., E39A). Importantly, it was observed that the fusion of twoother transactivation domains to the E2 DNA binding domain did notsuppress the growth of HeLa cells, even though each of these chimericproteins could function as E2-dependent transactivators in these cells.Therefore, some characteristic of the E2 transactivation domain isspecific for cell growth suppression and is not shared with othertransactivation domains. While not wishing to be bound by any particulartheory, it is possible that the E2 transactivation domain recruitsspecific cellular factors to the promoter which may play a role in thetranscriptional repression and growth suppression activities of E2.

The dependence of a functional transactivation domain may be in part dueto its ability to relieve nucleosome-mediated repression. The E2transactivation domain has been shown to counteract the nucleosomerepression of DNA replication (Li et al. (1994) PNAS, 91:7051-7055). Thetranscriptional repression properties of the E2-TR have beencharacterized on transiently transfected naked DNA. This, however,cannot be the only explanation for E2-mediated growth suppression, sincethe VP16 transactivation domain also has the ability to relievenucleosome repression, but in our experiments, expression of thechimeric protein does not result in growth suppression.

The growth-suppressive effect of E2 was demonstrated in otherHPV-positive cell lines but not in HPV-negative cell lines by thecotransfection assay used in this study. These results demonstrate thatE2 selectively represses cell growth in papillomavirus-infected cellsand not in uninfected cells. Importantly, these observations indicatethat the E2 polypeptides of the invention can be used therapeutically toselectivity arrest an undesired papillomavirus infections while leavingnormal cellular growth unaffected. This result contrasts with thefinding of Hwang et al. (Hwang et al. (1993) J. Virol., 67:3720-3729),who observed a growth-suppressive effect with BPV-1 E2-TA in theHPV-negative cervical carcinoma cell line HT-3. One explanation for thedifference between these two studies may involve the different assaysused and different levels of expression of E2 achieved in the twoexperimental approaches. Our studies were done with DNA transfection,whereas the studies by Hwang et al. used the recombinant SV40 virusPAVA-E2, which resulted in much higher levels of protein expression. Wehave not been able to detect E2 protein by immunoblot analysis in HeLacell lines transfected with the E2 plasmid, even though we can readilydetect functional levels of E2 in transactivation of transcriptionalrepression assays. However, E2 is readily detectable by immunoblottingHeLa cells infected with the PAVA-E2 virus (Hwang et al. (1993) J.Virol., 67:3720-3729), indicating a significantly higher level ofexpression. The two studies also differed in the experimental conditionsto assess the biologic effects of E2 expression. Hwang et al. examinedthe transient effects of E2 by analyzing the cells 2 days postinfection.In the colony growth suppression assay used in our study, selection wasmaintained for 2 weeks, at which time the number of drug-resistantcolonies was counted. Thus, the different effects of E2 observed on thegrowth of the HPV-negative HT-3 cell line may be due to differences inthe assays, to the levels of E2 expression achieved by the differentvectors, or to both. Nevertheless, it is the present findings which nowprovide the motivation to use E2 proteins to treat PV infected ortransformed cells.

Repression of HPV-18 E6/E7 transcription was observed in the HeLa-tsE2cells after a shift to the permissive temperature, resulting in a growtharrest. Our data indicate that in HeLa cells, the mechanism ofE2-mediated growth arrest involves a decrease in E6 and E7 mRNA andreactivation of the p53/p21 and pRB pathways. The fact that the p21/Cdkinhibitory pathway is still intact and can be activated by disruption ofE6 function suggests that therapeutic strategies targeted at interferingwith E6 function could be effective in inhibition of cellularproliferation in HPV-positive cancers. We also observe an accumulationof hypophosphorylated pRB, which may be a consequence of a p53-mediatedG₁ growth arrest (Slebos et al. (1994) Proc. Natl. Acad. Sci. USA,91:5320-5324). Cells blocked in G₁ prior to activation of the cyclinD/Cdk complexes do not phosphorylate pRB. The shift in thephosphorylation state of RB to its hypophosphorylated form suggests thatthe cell cycle block occurs prior to the point of pRB phosphorylation.Our studies are in agreement with the observation that expression ofantisense E6/E7 in C4-1, an HPV-18-derived cell line, was able toinhibit the growth rate of these cells (von Knebel-Doeberitz et al.(1988) Cancer Res., 48 :3780-3785; von Knebel-Doeberitz et al. (1992)Int. J. Cancer, 51:831-834).

The studies presented here indicate that the transactivation domain ofE2 is involved in the growth suppression and that the characteristic ofthe E2 transactivation domain in this suppression is not a generalproperty shared with the VP16 and Spi transactivation domains.

All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated polynucleotide encoding a functional portion of a humanpapillomavirus ad region and a functional portion of a humanpapillomavirus db region, wherein the encoded polypeptide comprises analtered hinge region modified by one or more amino acid substitutions,deletions, or additions as compared to wild type E2 polypeptide andselected for its ability to inhibit human papillomavirus DNA replicationand the growth of cells infected with a papillomavirus when expressed inthe cells, wherein the polynucleotide is present in a vector suitablefor expression in the infected cells.
 2. The isolated polynucleotide ofclaim 1, wherein the vector is a viral vector.
 3. The isolatedpolynucleotide of claim 2, wherein the vector is an adenoviral vector.4. A composition comprising the polynucleotide of any one of claims 1, 2or 3 and a pharmaceutically acceptable carrier.
 5. The polynucleotide ofclaim 1, wherein the human papillomavirus (HPV) sequence is selectedfrom the group consisting of HPV-16, HPV-18, HPV-31, and HPV-33.
 6. Anisolated polynucleotide encoding an E2_(ad/db) polypeptide, wherein theE2_(ad/db) polypeptide comprises an altered hinge region modified by oneor more amino acid substitutions, deletions, or additions as compared towild type E2 polypeptide and selected for its ability to inhibitpapillomavirus DNA replication and the growth of cells infected with apapillomavirus when expressed in the cells.
 7. A composition comprisingthe polynucleotide of claim 6 and a pharmaceutically acceptable carrier.8. The polynucleotide of claim 6, wherein the papillomavirus is selectedfrom the group consisting of HPV-16, HPV-18, HPV-31, and HPV-33.
 9. Theisolated polynucleotide of claim 1, wherein the E2_(ad/db) polypeptidecomprises a deletion corresponding to amino acid residues 220-309 of BPVE2.
 10. The isolated polynucleotide of claim 6, wherein the E2_(ad/db)polypeptide comprises a deletion corresponding to amino acid residues220-309 of BPV E2.